Isomerases, nucleic acids encoding them and methods for making and using them

ABSTRACT

This invention relates generally to enzymes, polynucleotides encoding the enzymes having isomerase activity, e.g., racemase activity, e.g., amino acid racemase activity, alanine racemase activity, and/or epimerase activity, and/or catalyze the re-arrangement of atoms within a molecule, catalyze the conversion of one isomer into another, catalyze the conversion of an optically active substrate into a raceme, which is optically inactive, catalyze the interconversion of substrate enantiomers, catalyze the stereochemical inversion around the asymmetric carbon atom in a substrate having only one center of asymmetry, catalyze the stereochemical inversion of the configuration around an asymmetric carbon atom in a substrate having more than one asymmetric center, and/or catalyze the racemization of amino acids.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/810,067, filed Sep. 30, 2010, now U.S. Pat. No. 8,541,220; which is anational stage application claiming benefit of priority under 35 U.S.C.§371 of International Application number PCT/US2008/088066 having aninternational filing date of Dec. 22, 2008, and published as WO2009/088066, on Jul. 16, 2009, which claims the benefit of priorityunder 35 U.S.C. §119(e) to Provisional Application No. 61/018,880 filedJan. 3, 2008. The contents of the above mentioned patent applicationsare incorporated by reference herein in their entirety and for allpurposes.

REFERENCE TO SEQUENCE LISTING SUBMITTED VIA EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP §502.05(IX), is incorporated herein by reference in itsentirety for all purposes. The sequence listing is identified on theelectronically filed .txt file as follows:

File Name Date of Creation Size SEQUENCELISTINGD244001ND1 Nov. 27, 20131.25 MB (1,311,493 bytes)

FIELD OF THE INVENTION

This invention relates generally to enzymes, polynucleotides encodingthe enzymes, the use of such polynucleotides and polypeptides and morespecifically to enzymes having isomerase activity, e.g., racemaseactivity, e.g., amino acid racemase activity, alanine racemase activity,and/or epimerase activity, and/or catalyze the re-arrangement of atomswithin a molecule, catalyze the conversion of one isomer into another,catalyze the conversion of an optically active substrate into a raceme,which is optically inactive, catalyze the interconversion of substrateenantiomers, catalyze the stereochemical inversion around the asymmetriccarbon atom in a substrate having only one center of asymmetry, catalyzethe stereochemical inversion of the configuration around an asymmetriccarbon atom in a substrate having more than one asymmetric center,and/or catalyze the racemization of amino acids. Thus, the inventionprovides enzymes, compositions, methods for production of pharmaceutical(drug) compositions, pharmaceutical (drug) precursors and intermediates,antibiotics, sweeteners, peptide enzymes, peptide hormones, fuel andfuel additive compositions, foods and food additives, beverage andbeverage additives, feeds and feed additives, drugs and drug additives,dietary supplements, textiles, wood, paper, pulp, and detergentscomprising the polypeptides or polynucleotides in accordance with theinvention.

BACKGROUND

Isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases catalyze the re-arrangement of atoms withina molecule, catalyze the conversion of one isomer into another, catalyzethe conversion of an optically active substrate into a raceme, which isoptically inactive, catalyze the interconversion of substrateenantiomers, catalyze the stereochemical inversion around the asymmetriccarbon atom in a substrate having only one center of asymmetry, catalyzethe stereochemical inversion of the configuration around an asymmetriccarbon atom in a substrate having more than one asymmetric center,and/or catalyze the racemization of amino acids. Isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases are of considerable commercial value, being used in thepharmaceutical industry, in the food, feed and beverage industries, e.g.for the production of sweeteners, in the wood/paper industry and in thefuel industry.

SUMMARY OF THE INVENTION

This invention provides enzymes having isomerase activity, e.g.,racemase activity, e.g., amino acid racemase activity, alanine racemaseactivity, and/or epimerase activity, and/or catalyze the re-arrangementof atoms within a molecule, catalyze the conversion of one isomer intoanother, catalyze the conversion of an optically active substrate into araceme, which is optically inactive, catalyze the interconversion ofsubstrate enantiomers, catalyze the stereochemical inversion around theasymmetric carbon atom in a substrate having only one center ofasymmetry, catalyze the stereochemical inversion of the configurationaround an asymmetric carbon atom in a substrate having more than oneasymmetric center, and/or catalyze the racemization of amino acids. Theinvention further provides enzymes having isomerase activity, e.g.,racemase activity, e.g., amino acid racemase activity, alanine racemaseactivity, and/or epimerase activity and nucleic acids encoding them,vectors and cells comprising them, probes for amplifying and identifyingthese an isomerase-, e.g., a racemase-, e.g., an amino acid racemase-,an alanine racemase-, and/or an epimerase-isomerase-, e.g., racemase-,e.g., amino acid racemase-, alanine racemase-, and/or epimerase-encodingnucleic acids, and methods for making and using these polypeptides andpeptides.

The invention provides enzymes, compositions, methods for production ofpharmaceutical (drug) compositions, pharmaceutical (drug) precursors andintermediates, antibiotics, sweeteners, peptide enzymes, peptidehormones, fuel and fuel additive compositions, foods and food additives,beverage and beverage additives, feeds and feed additives, drugs anddrug additives, dietary supplements, textiles, wood, paper, pulp, anddetergents comprising the polypeptides or polynucleotides in accordancewith the invention. These compositions can be formulated in a variety offorms, such as tablets, gels, pills, implants, liquids, sprays, films,micelles, powders, food, feed pellets or as any type of encapsulatedform.

In some embodiments, the isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases and/or compositionsthereof may be useful in pharmaceutical, industrial, and/or agriculturalcontexts.

In some embodiments, the isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases and/or compositionsthereof may be useful for catalyzing the inversion of stereochemistry inbiological molecules. In some embodiments, the isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases and/or compositions thereof may be useful for catalyzing theinterconversion of substrate enantiomers. In some embodiments,isomerases, e.g., racemases, e.g., amino acid racemases, and/or alanineracemases catalyze the stereochemical inversion around the asymmetriccarbon atom in a substrate having only one center of asymmetry. In someembodiments, isomerases, e.g., epimerases catalyze the stereochemicalinversion of the configuration around an asymmetric carbon atom in asubstrate having more than one asymmetric center. In some embodiments,isomerases, e.g., racemases, e.g., amino acid racemases, and/or alanineracemases are provided that catalyze the racemization of amino acids. Insome embodiments, racemases are provided that catalyze the racemizationof a specific amino acid. In some embodiments, isomerases, e.g.,racemases, e.g., amino acid racemases, and/or alanine racemases areprovided that catalyze the racemization of several amino acids.

In some embodiments, the racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases and/or compositions thereof may be usefulin D-amino acid metabolism. D-amino acids are necessary for bacterialgrowth and for peptidoglycan assembly and cross linking. D-amino acidsare also present in the brains of newborn humans. Bacterial serineracemase plays and important role in vancomycin resistance. Some aminoacid racemases are PLP dependent; other amino acid racemases are PLPindependent. (see, e.g., Yoshimura, T., N. Esaki, 2003, Journal ofBioscience and Bioengineering. 96:103-109). In alternative embodiments,the racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases and/or combinations thereof are components in pharmaceutical(drug) compositions, pharmaceutical (drug) precursors and/orintermediates, e.g. as antibiotics or for treatment of amino aciddeficiencies.

In alternative embodiments, the isomerases, e.g., racemases, e.g., aminoacid racemases, alanine racemases, and/or epimerases of the inventionand/or compositions thereof of may be useful as an antibiotic or in thepreparation of antibiotics (see, e.g., Strych, U., M. J. Benedik. 2002,Journal of Bacteriology. 184:4321-4325).

In alternative embodiments, the isomerases, e.g., racemases, e.g., aminoacid racemases, alanine racemases, and/or epimerases of the inventionand/or compositions thereof of may be useful as in mediation of mammalnervous transmission and maintenance of bacterial cell wall rigidity andstrength (see, e.g. Liu, L., K. Iwata, M. Yohda, K. Mild. 2002, FEBS.528:114-118).

In alternative embodiments, the invention provides enzymes and processesfor the bioconversion of any biomass into fuel, e.g. biofuel, e.g.,ethanol, propanol, butanol, methanol, and/or biodiesel or biofuels suchas synthetic liquids or gases, such as syngas, and the production ofother fermentation products, e.g. succinic acid, lactic acid, or aceticacid.

In alternative embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having at least one conservative aminoacid substitution and retaining its isomerase activity, e.g., racemaseactivity, e.g., amino acid racemase activity, alanine racemase activity,and/or epimerase activity; or, wherein the at least one conservativeamino acid substitution comprises substituting an amino acid withanother amino acid of like characteristics; or, a conservativesubstitution comprises: replacement of an aliphatic amino acid withanother aliphatic amino acid; replacement of a Serine with a Threonineor vice versa; replacement of an acidic residue with another acidicresidue; replacement of a residue bearing an amide group with anotherresidue bearing an amide group; exchange of a basic residue with anotherbasic residue; or replacement of an aromatic residue with anotheraromatic residue;

In alternative embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity but lacking a signalsequence, a prepro domain and/or other domain.

In alternative embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity further comprising aheterologous sequence; and in one aspect, the heterologous sequencecomprises, or consists of a sequence encoding: (i) a heterologous signalsequence, a heterologous domain, a heterologous dockerin domain, aheterologous catalytic domain (CD), or a combination thereof; (ii) thesequence of (i), wherein the heterologous signal sequence, domain orcatalytic domain (CD) is derived from a heterologous enzyme; or, (iii) atag, an epitope, a targeting peptide, a cleavable sequence, a detectablemoiety or an enzyme; and in one aspect, the heterologous signal sequencetargets the encoded protein to a vacuole, the endoplasmic reticulum, achloroplast or a starch granule.

In alternative embodiments, the invention provides polypeptides (and thenucleic acids that encode them) having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity, wherein thepolypeptides are cofactor dependent or cofactor independent. In oneembodiment, a cofactor dependent polypeptide requires the presence of anon-protein component to be functional. In one embodiment, the cofactorcomprises a metal ion, a coenzyme, a pyridoxal-phosphate and or aphosphopantetheine.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising (a) a nucleic acid (polynucleotide) encoding at least onepolypeptide, wherein the nucleic acid comprises a sequence having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or complete(100%) sequence identity to the nucleic acid (polynucleotide) sequenceof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ IDNO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203,SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ IDNO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231,SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ IDNO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259,SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ IDNO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287,SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315,SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ IDNO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343,SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ IDNO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371,SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379, SEQ IDNO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQID NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399,SEQ ID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ IDNO:409, SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQID NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427,SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, SEQ IDNO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455,SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ IDNO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQID NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ ID NO:483,SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491, SEQ IDNO:493, SEQ ID NO:495 or SEQ ID NO:497; wherein the nucleic acid encodesat least one polypeptide having an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity, or encodes a polypeptide orpeptide capable of generating an isomerase specific antibody, e.g., aracemase specific antibody, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase specific antibody (a polypeptide orpeptide that acts as an epitope or immunogen),

(b) the nucleic acid (polynucleotide) of (a), wherein the sequenceidentities are determined: (A) by analysis with a sequence comparisonalgorithm or by a visual inspection, or (B) over a region of at leastabout 20, 30, 40, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 ormore residues, or over the full length of a cDNA, transcript (mRNA) orgene;

(c) the nucleic acid (polynucleotide) of (a) or (b), wherein thesequence comparison algorithm is a BLAST version 2.2.2 algorithm where afiltering setting is set to blastall-p blastp-d “nr pataa”-F F, and allother options are set to default;

(d) a nucleic acid (polynucleotide) encoding at least one polypeptide orpeptide,

wherein the nucleic acid comprises a sequence that hybridizes understringent conditions to a nucleic acid comprising the sequence of SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31,SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41,SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51,SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61,SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ ID NO:71,SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ ID NO:81,SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ ID NO:91,SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ ID NO:101,SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQ IDNO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119, SEQID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129,SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ IDNO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ ID NO:157,SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQ IDNO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175, SEQID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ ID NO:185,SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQ IDNO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203, SEQID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ ID NO:213,SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQ IDNO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231, SEQID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ ID NO:241,SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQ IDNO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269,SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQ IDNO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287, SEQID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ ID NO:297,SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQ IDNO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315, SEQID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ ID NO:325,SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQ IDNO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343, SEQID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ ID NO:353,SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQ IDNO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371, SEQID NO:373, SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379, SEQ ID NO:381,SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQ IDNO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399, SEQID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ ID NO:409,SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQ IDNO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427, SEQID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, SEQ ID NO:437,SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQ IDNO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455, SEQID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ ID NO:465,SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQ IDNO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ ID NO:483, SEQID NO:485, SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491, SEQ ID NO:493,SEQ ID NO:495 or SEQ ID NO:497,

and the stringent conditions comprise a wash step comprising a wash in0.2×SSC at a temperature of about 65° C. for about 15 minutes;

(e) the nucleic acid (polynucleotide) of any of (a) to (d) having alength of at least about 20, 25, 30, 50, 75, 100, 125, 150, 175, 200,225, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1050, 1100, 1150 or more nucleotide residues, or the fulllength of a gene or a transcript;

(f) a nucleic acid (polynucleotide) encoding at least one polypeptidehaving an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity, wherein the polypeptide comprises the sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ IDNO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ IDNO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ IDNO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ IDNO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ IDNO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ IDNO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ IDNO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ IDNO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120,SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ IDNO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148,SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ IDNO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176,SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ IDNO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204,SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ IDNO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232,SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ IDNO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260,SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ IDNO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288,SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ IDNO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316,SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ IDNO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344,SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ IDNO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372,SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ IDNO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQID NO:392, SEQ ID NO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400,SEQ ID NO:402, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ IDNO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQID NO:420, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428,SEQ ID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ IDNO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQID NO:448, SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456,SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ IDNO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQID NO:476, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484,SEQ ID NO:486, SEQ ID NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ IDNO:494, SEQ ID NO:496 or SEQ ID NO:498, or enzymatically activefragments thereof;

(g) the nucleic acid (polynucleotide) of any of (a) to (f) and encodinga polypeptide having at least one conservative amino acid substitutionand retaining its isomerase activity, e.g., racemase activity, e.g.,amino acid racemase activity, alanine racemase activity, and/orepimerase activity, wherein the at least one conservative amino acidsubstitution comprises substituting an amino acid with another aminoacid of like characteristics; or, a conservative substitution comprises:replacement of an aliphatic amino acid with another aliphatic aminoacid; replacement of a Serine with a Threonine or vice versa;replacement of an acidic residue with another acidic residue;replacement of a residue bearing an amide group with another residuebearing an amide group; exchange of a basic residue with another basicresidue; or replacement of an aromatic residue with another aromaticresidue;

(h) the nucleic acid (polynucleotide) of any of (a) to (g) encoding apolypeptide having an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity but lacking a signal sequence, a preprodomain, and/or other domain;

(i) the nucleic acid (polynucleotide) of any of (a) to (h) encoding apolypeptide having an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity further comprising a heterologous sequence;

(j) the nucleic acid (polynucleotide) of (i), wherein the heterologoussequence comprises, or consists of a sequence encoding: (A) aheterologous signal sequence, a heterologous domain, a heterologousdockerin domain, a heterologous catalytic domain (CD), or a combinationthereof; (B) the sequence of (i), wherein the heterologous signalsequence, domain or catalytic domain (CD) is derived from a heterologousenzyme; or, (C) a tag, an epitope, a targeting peptide, a cleavablesequence, a detectable moiety or an enzyme;

(k) the nucleic acid (polynucleotide) of (j), wherein the heterologoussignal sequence targets the encoded protein to a vacuole, theendoplasmic reticulum, a chloroplast or a starch granule; or

(l) a nucleic acid sequence (polynucleotide) fully (completely)complementary to the sequence of any of (a) to (k).

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid encoding at least one polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, wherein the polypeptide has a sequence as set forth in SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ IDNO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158,SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186,SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ IDNO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214,SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ IDNO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242,SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ IDNO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270,SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ IDNO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298,SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ IDNO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326,SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ IDNO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354,SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ IDNO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQID NO:374, SEQ ID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382,SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ IDNO:392, SEQ ID NO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQID NO:402, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410,SEQ ID NO:412, SEQ ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ IDNO:420, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438,SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ IDNO:448, SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466,SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ IDNO:476, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQID NO:486, SEQ ID NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494,SEQ ID NO:496 or SEQ ID NO:498, or enzymatically active fragmentsthereof, including the sequences described herein and in Tables 1, 2 and3, and the Sequence Listing (all of these sequences are “exemplaryenzymes/polypeptides of the invention”), and enzymatically activesubsequences (fragments) thereof and/or immunologically activesubsequences thereof (such as epitopes or immunogens) (all “peptides ofthe invention”) and variants thereof (all of these sequencesencompassing polypeptide and peptide sequences of the invention) (or,hereinafter referred to as the exemplary polypeptide sequences of theinventions).

The invention provides isolated, synthetic or recombinant nucleic acidscomprising sequences completely complementary to all of these nucleicacid sequences of the invention (complementary (non-coding) and codingsequences also hereinafter collectively referred to as nucleic acidsequences of the invention).

In one aspect, the sequence identity is at least about 51%, 52%, 53%,54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or 100% (complete) sequence identity (homology). Inone aspect, the sequence identity is over a region of at least about150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150 or more residues,or the full length of a gene or a transcript. For example, the inventionprovides isolated, synthetic or recombinant nucleic acids comprising anucleic acid sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ IDNO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ IDNO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ IDNO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ IDNO:67, SEQ ID NO:69, SEQ ID NO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ IDNO:77, SEQ ID NO:79, SEQ ID NO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ IDNO:87, SEQ ID NO:89, SEQ ID NO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ IDNO:97, SEQ ID NO:99, SEQ ID NO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ IDNO:107, SEQ ID NO:109, SEQ ID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQID NO:117, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125,SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ IDNO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQID NO:145, SEQ ID NO:147, SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153,SEQ ID NO:155, SEQ ID NO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ IDNO:163, SEQ ID NO:165, SEQ ID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQID NO:173, SEQ ID NO:175, SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181,SEQ ID NO:183, SEQ ID NO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ IDNO:191, SEQ ID NO:193, SEQ ID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQID NO:201, SEQ ID NO:203, SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209,SEQ ID NO:211, SEQ ID NO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ IDNO:219, SEQ ID NO:221, SEQ ID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQID NO:229, SEQ ID NO:231, SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237,SEQ ID NO:239, SEQ ID NO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ IDNO:247, SEQ ID NO:249, SEQ ID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265,SEQ ID NO:267, SEQ ID NO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ IDNO:275, SEQ ID NO:277, SEQ ID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQID NO:285, SEQ ID NO:287, SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293,SEQ ID NO:295, SEQ ID NO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ IDNO:303, SEQ ID NO:305, SEQ ID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQID NO:313, SEQ ID NO:315, SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321,SEQ ID NO:323, SEQ ID NO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ IDNO:331, SEQ ID NO:333, SEQ ID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQID NO:341, SEQ ID NO:343, SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349,SEQ ID NO:351, SEQ ID NO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ IDNO:359, SEQ ID NO:361, SEQ ID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQID NO:369, SEQ ID NO:371, SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377,SEQ ID NO:379, SEQ ID NO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ IDNO:387, SEQ ID NO:389, SEQ ID NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQID NO:397, SEQ ID NO:399, SEQ ID NO:401, SEQ ID NO:403, SEQ ID NO:405,SEQ ID NO:407, SEQ ID NO:409, SEQ ID NO:411, SEQ ID NO:413, SEQ IDNO:415, SEQ ID NO:417, SEQ ID NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQID NO:425, SEQ ID NO:427, SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433,SEQ ID NO:435, SEQ ID NO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ IDNO:443, SEQ ID NO:445, SEQ ID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQID NO:453, SEQ ID NO:455, SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461,SEQ ID NO:463, SEQ ID NO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ IDNO:471, SEQ ID NO:473, SEQ ID NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQID NO:481, SEQ ID NO:483, SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:489,SEQ ID NO:491, SEQ ID NO:493, SEQ ID NO:495 or SEQ ID NO:497, e.g., asdescribed in Tables 1, 2 and 3 and in the Sequence Listing (all of thesesequences are “exemplary p of the invention”), and enzymatically activesubsequences (fragments) thereof.

The invention provides isolated, synthetic or recombinant nucleic acidsencoding a polypeptide having an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity, wherein the nucleic acid has atleast one sequence modification of an exemplary sequence of theinvention, or, any sequence of the invention.

In one aspect (optionally), the isolated, synthetic or recombinantnucleic acids of the invention have an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity, e.g., wherein theactivity comprises catalyzing the re-arrangement of atoms within amolecule, catalyzing the conversion of one isomer into another,catalyzing the conversion of an optically active substrate into araceme, which is optically inactive, catalyzing the interconversion ofsubstrate enantiomers, catalyzing the stereochemical inversion aroundthe asymmetric carbon atom in a substrate having only one center ofasymmetry, catalyzing the stereochemical inversion of the configurationaround an asymmetric carbon atom in a substrate having more than oneasymmetric center, and/or catalyzing the racemization of amino acids.

In one aspect, the isomerase activity, e.g., racemase activity, e.g.,amino acid racemase activity, alanine racemase activity, and/orepimerase activity is thermostable, e.g., wherein the polypeptideretains an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity under conditions comprising a temperature range fromabout −100° C. to about −80° C., about −80° C. to about −40° C., about−40° C. to about −20° C., about −20° C. to about 0° C., about 0° C. toabout 5° C., about 5° C. to about 15° C., about 15° C. to about 25° C.,about 25° C. to about 37° C., about 37° C. to about 45° C., about 45° C.to about 55° C., about 55° C. to about 70° C., about 70° C. to about 75°C., about 75° C. to about 85° C., about 85° C. to about 90° C., about90° C. to about 95° C., about 95° C. to about 100° C., about 100° C. toabout 105° C., about 105° C. to about 110° C., about 110° C. to about120° C., or 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C.,102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C.,110° C., 111° C., 112° C., 113° C., 114° C., 115° C. or more. In someembodiments, the thermostable polypeptides according to the inventionretains activity, e.g., an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity, at a temperature in the rangesdescribed above, at about pH 3.0, about pH 3.5, about pH 4.0, about pH4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, about pH7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9.0, about pH9.5, about pH 10.0, about pH 10.5, about pH 11.0, about pH 11.5, aboutpH 12.0 or more.

In one aspect, the isomerase activity, e.g., racemase activity, e.g.,amino acid racemase activity, alanine racemase activity, and/orepimerase activity is thermotolerant, e.g., wherein the polypeptideretains an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity after exposure to a temperature in the range fromabout −100° C. to about −80° C., about −80° C. to about −40° C., about−40° C. to about −20° C., about −20° C. to about 0° C., about 0° C. toabout 5° C., about 5° C. to about 15° C., about 15° C. to about 25° C.,about 25° C. to about 37° C., about 37° C. to about 45° C., about 45° C.to about 55° C., about 55° C. to about 70° C., about 70° C. to about 75°C., about 75° C. to about 85° C., about 85° C. to about 90° C., about90° C. to about 95° C., about 95° C. to about 100° C., about 100° C. toabout 105° C., about 105° C. to about 110° C., about 110° C. to about120° C., or 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C.,102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C.,110° C., 111° C., 112° C., 113° C., 114° C., 115° C. or more. Thethermotolerant polypeptides according to the invention can retainactivity, e.g. an isomerase activity, e.g., a racemase activity, e.g.,an amino acid racemase activity, an alanine racemase activity, and/or anepimerase activity, after exposure to a temperature in the range fromabout −100° C. to about −80° C., about −80° C. to about −40° C., about−40° C. to about −20° C., about −20° C. to about 0° C., about 0° C. toabout 5° C., about 5° C. to about 15° C., about 15° C. to about 25° C.,about 25° C. to about 37° C., about 37° C. to about 45° C., about 45° C.to about 55° C., about 55° C. to about 70° C., about 70° C. to about 75°C., about 75° C. to about 85° C., about 85° C. to about 90° C., about90° C. to about 95° C., about 95° C. to about 100° C., about 100° C. toabout 105° C., about 105° C. to about 110° C., about 110° C. to about120° C., or 95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C.,102° C., 103° C., 104° C., 105° C., 106° C., 107° C., 108° C., 109° C.,110° C., 111° C., 112° C., 113° C., 114° C., 115° C. or more. In someembodiments, the thermotolerant polypeptides according to the inventionretains activity, e.g. an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity, after exposure to a temperature in theranges described above, at about pH 3.0, about pH 3.5, about pH 4.0,about pH 4.5, about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5,about pH 7.0, about pH 7.5, about pH 8.0, about pH 8.5, about pH 9.0,about pH 9.5, about pH 10.0, about pH 10.5, about pH 11.0, about pH11.5, about pH 12.0 or more.

In one aspect, the isomerase activity, e.g., racemase activity, e.g.,amino acid racemase activity, alanine racemase activity, and/orepimerase activity of polypeptides encoded by nucleic acids of theinvention retain activity under acidic conditions comprising about pH6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4.0, pH 3.5, pH 3.0 or less (moreacidic) pH, or, retain an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity after exposure to acidic conditionscomprising about pH 6.5, pH 6, pH 5.5, pH 5, pH 4.5, pH 4.0, pH 3.5, pH3.0 or less (more acidic) pH; or, retain activity under basic conditionscomprising about pH 7, pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more basic) or, retain anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity after exposure to basic conditions comprising about pH 7, pH7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10, pH 10.5, pH 11, pH 11.5, pH 12,pH 12.5 or more (more basic). In one aspect, isomerase activity, e.g.,racemase activity, e.g., amino acid racemase activity, alanine racemaseactivity, and/or epimerase activity of polypeptides encoded by nucleicacids of the invention retain activity at a temperature of at leastabout 80° C., 81° C., 82° C., 83° C., 84° C., 85° C., 86° C., 87° C.,88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C., 95° C., 96° C.,97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103° C., 103.5° C.,104° C., 105° C., 107° C., 108° C., 109° C. or 110° C., or more, and abasic pH of at least about pH 7.5 pH 8.0, pH 8.5, pH 9, pH 9.5, pH 10,pH 10.5, pH 11, pH 11.5, pH 12, pH 12.5 or more (more basic).

The invention provides expression cassettes, cloning vehicles, or avector (e.g., expression vectors) comprising a nucleic acid comprising asequence of the invention. The cloning vehicle can comprise a viralvector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, abacteriophage or an artificial chromosome. The viral vector can comprisean adenovirus vector, a retroviral vector or an adeno-associated viralvector. The cloning vehicle can comprise an artificial chromosomecomprising a bacterial artificial chromosome (BAC), a bacteriophageP1-derived vector (PAC), a yeast artificial chromosome (YAC), or amammalian artificial chromosome (MAC).

The invention provides nucleic acid probes for identifying a nucleicacid encoding a polypeptide with an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity, wherein the probe comprises atleast about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 100,125, 150, 175, 200, 225, 250, 275, 300 or more consecutive bases of anucleic acid comprising an exemplary sequence of the invention, or, anysequence of the invention (as defined herein), wherein in one aspect(optionally) the probe comprises an oligonucleotide comprising betweenat least about 10 to 300, about 25 to 250, about 10 to 50, about 20 to60, about 30 to 70, about 40 to 80, about 60 to 100, or about 50 to 150or more consecutive bases.

The invention provides amplification primer pairs for amplifying anucleic acid encoding a polypeptide having an isomerase activity, e.g.,a racemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity, wherein the primer pairis capable of amplifying a nucleic acid comprising an exemplary sequenceof the invention, or, any sequence of the invention (as defined herein),or a subsequence thereof, wherein optionally a member of theamplification primer sequence pair comprises an oligonucleotidecomprising at least about 10 to 50 consecutive bases of the sequence,or, about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive bases ofthe sequence. The invention provides amplification primer pairs whereinthe primer pair comprises a first member having a sequence as set forthby about the first (the 5′) 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or moreresidues of an exemplary sequence of the invention, or, any sequence ofthe invention (as defined herein), and a second member having a sequenceas set forth by about the first (the 5′) 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35or more residues of the complementary strand of the first member.

The invention provides an isomerase-, e.g., a racemase-, e.g., an aminoacid racemase-, an alanine racemase-, and/or an epimerase-encodingnucleic acids generated by amplification of a polynucleotide using anamplification primer pair of the invention, wherein optionally theamplification is by polymerase chain reaction (PCR). In one aspect, thenucleic acid is generated by amplification of a gene library, wherein inone aspect (optionally) the gene library is an environmental library.The invention provides isolated, synthetic or recombinant isomerases,e.g., racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases encoded by an isomerase-, e.g., a racemase-, e.g., an aminoacid racemase-, an alanine racemase-, and/or an epimerase-encodingnucleic acid generated by amplification of a polynucleotide using anamplification primer pair of the invention. The invention providesmethods of amplifying a nucleic acid encoding a polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, the methods comprising the step of amplification of a templatenucleic acid with an amplification primer sequence pair capable ofamplifying an exemplary sequence of the invention, or, any sequence ofthe invention (as defined herein), or a subsequence thereof.

The invention provides expression cassette, a vector or a cloningvehicle comprising a nucleic acid comprising a sequence of theinvention, wherein optionally the cloning vehicle comprises a viralvector, a plasmid, a phage, a phagemid, a cosmid, a fosmid, abacteriophage or an artificial chromosome. The viral vector can comprisean adenovirus vector, a retroviral vector or an adeno-associated viralvector, or, the artificial chromosome comprises a bacterial artificialchromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeastartificial chromosome (YAC), or a mammalian artificial chromosome (MAC).

The invention provides transformed cells comprising a nucleic acid orvector of the invention, or an expression cassette or cloning vehicle ofthe invention. The transformed cell can be a bacterial cell, a mammaliancell, a fungal cell, a yeast cell, an insect cell or a plant cell.

The invention provides transgenic non-human animals comprising asequence of the invention. The transgenic non-human animal can be amouse, a rat, a rabbit, a sheep, a pig, a chicken, a goat, a fish, adog, or a cow. The invention provides transgenic plants comprising asequence of the invention, e.g., wherein the plant is a corn plant, asorghum plant, a potato plant, a tomato plant, a wheat plant, an oilseedplant, a rapeseed plant, a soybean plant, a rice plant, a barley plant,a grass, or a tobacco plant. The invention provides transgenic seedscomprising a sequence of the invention, e.g., wherein the seed is a cornseed, a wheat kernel, an oilseed, a rapeseed, a soybean seed, a palmkernel, a sunflower seed, a sesame seed, a rice, a barley, a peanut or atobacco plant seed.

The invention provides antisense oligonucleotides comprising a nucleicacid sequence complementary to or capable of hybridizing under stringentconditions to a sequence of the invention (including, e.g., exemplarysequences of the invention), or a subsequence thereof, whereinoptionally the antisense oligonucleotide is between about 10 to 50,about 20 to 60, about 30 to 70, about 40 to 80, or about 60 to 100 basesin length, and in one aspect (optionally) the stringent conditionscomprise a wash step comprising a wash in 0.2×SSC at a temperature ofabout 65° C. for about 15 minutes.

The invention provides methods of inhibiting the translation of anisomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase message in a cell comprising administeringto the cell or expressing in the cell an antisense oligonucleotidecomprising a nucleic acid sequence complementary to or capable ofhybridizing under stringent conditions to a sequence of the invention(including, e.g., exemplary sequences of the invention).

The invention provides double-stranded inhibitory RNA (RNAi) moleculescomprising a subsequence of a sequence of the invention (including,e.g., exemplary sequences of the invention). The double-strandedinhibitory RNA (RNAi) molecule can be about 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 or more duplexnucleotides in length. The invention provides methods of inhibiting theexpression of an isomerase, e.g., a racemase, e.g., an amino acidracemase, an alanine racemase, and/or an epimerase in a cell comprisingadministering to the cell or expressing in the cell a double-strandedinhibitory RNA (iRNA), wherein the RNA comprises a subsequence of asequence of the invention (including, e.g., exemplary sequences of theinvention).

The invention provides isolated, synthetic or recombinant polypeptideshaving an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity, or polypeptides capable of generating an immuneresponse specific for an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase (e.g., anepitope); and in alternative aspects peptide and polypeptide of theinvention comprise a sequence:

(a) comprising an amino acid sequence having at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or has 100% (complete) sequenceidentity to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ IDNO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ IDNO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ IDNO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ IDNO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ IDNO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ IDNO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ IDNO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118,SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ IDNO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146,SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ IDNO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174,SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ IDNO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202,SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ IDNO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230,SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ IDNO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258,SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ IDNO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286,SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ IDNO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314,SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ IDNO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342,SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ IDNO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370,SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376, SEQ ID NO:378, SEQ IDNO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQ ID NO:396, SEQ ID NO:398,SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404, SEQ ID NO:406, SEQ IDNO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ ID NO:414, SEQ ID NO:416, SEQID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426,SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ IDNO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454,SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ IDNO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:482,SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488, SEQ ID NO:490, SEQ IDNO:492, SEQ ID NO:494, SEQ ID NO:496 or SEQ ID NO:498, or enzymaticallyactive fragments thereof,

wherein the polypeptide or peptide of (i) or (ii) has an isomeraseactivity, e.g., a racemase activity, e.g., an amino acid racemaseactivity, an alanine racemase activity, and/or an epimerase activity, orthe polypeptide or peptide is capable of generating an isomerasespecific antibody, e.g., a racemase specific antibody, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase specificantibody (a polypeptide or peptide that acts as an epitope orimmunogen),

(b) the polypeptide or peptide of (a), wherein the sequence identitiesare determined: (A) by analysis with a sequence comparison algorithm orby a visual inspection, or (B) over a region of at least about 20, 25,30, 35, 40, 45, 50, 55, 60, 75, 100, 150, 200, 250, 300 or more aminoacid residues, or over the full length of the polypeptide or peptide orenzyme, and/or enzymatically active subsequences (fragments) thereof,

(c) the polypeptide or peptide of (a) of (b), wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by a visual inspection, and optionally the sequencecomparison algorithm is a BLAST version 2.2.2 algorithm where afiltering setting is set to blastall-p blastp-d “nr pataa”-F F, and allother options are set to default;

(d) an amino acid sequence encoded by the nucleic acid of claim 1,wherein the polypeptide has (i) an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity, or, (ii) has immunogenicactivity in that it is capable of generating an antibody thatspecifically binds to a polypeptide having a sequence of (a), and/orenzymatically active subsequences (fragments) thereof;

(e) the amino acid sequence of any of (a) to (d), and comprising atleast one amino acid residue conservative substitution, and thepolypeptide or peptide retains isomerase activity, e.g., racemaseactivity, e.g., amino acid racemase activity, alanine racemase activity,and/or epimerase activity;

(e) the amino acid sequence of (d), wherein the conservativesubstitution comprises replacement of an aliphatic amino acid withanother aliphatic amino acid; replacement of a serine with a threonineor vice versa; replacement of an acidic residue with another acidicresidue; replacement of a residue bearing an amide group with anotherresidue bearing an amide group; exchange of a basic residue with anotherbasic residue; or, replacement of an aromatic residue with anotheraromatic residue, or a combination thereof,

(f) the amino acid sequence of (e), wherein the aliphatic residuecomprises Alanine, Valine, Leucine, Isoleucine or a synthetic equivalentthereof; the acidic residue comprises Aspartic acid, Glutamic acid or asynthetic equivalent thereof; the residue comprising an amide groupcomprises Aspartic acid, Glutamic acid or a synthetic equivalentthereof; the basic residue comprises Lysine, Arginine or a syntheticequivalent thereof; or, the aromatic residue comprises Phenylalanine,Tyrosine or a synthetic equivalent thereof;

(g) the polypeptide of any of (a) to (f) having an isomerase activity,e.g., a racemase activity, e.g., an amino acid racemase activity, analanine racemase activity, and/or an epimerase activity but lacking asignal sequence, a prepro domain, and/or other domain,

(h) the polypeptide of any of (a) to (g) having an isomerase activity,e.g., a racemase activity, e.g., an amino acid racemase activity, analanine racemase activity, and/or an epimerase activity furthercomprising a heterologous sequence;

(i) the polypeptide of (h), wherein the heterologous sequence comprises,or consists of: (A) a heterologous signal sequence, a heterologousdomain, a heterologous dockerin domain, a heterologous catalytic domain(CD), or a combination thereof; (B) the sequence of (A), wherein theheterologous signal sequence, domain or catalytic domain (CD) is derivedfrom a heterologous enzyme; and/or, (C) a tag, an epitope, a targetingpeptide, a cleavable sequence, a detectable moiety or an enzyme;

(j) polypeptide of (i), wherein the heterologous signal sequence targetsthe encoded protein to a vacuole, the endoplasmic reticulum, achloroplast or a starch granule; or

(k) comprising an amino acid sequence encoded any nucleic acid sequenceof this invention.

In one aspect, the isomerase activity, e.g., racemase activity, e.g.,amino acid racemase activity, alanine racemase activity, and/orepimerase activity comprises catalyzing the re-arrangement of atomswithin a molecule, catalyzing the conversion of one isomer into another,catalyzing the conversion of an optically active substrate into araceme, which is optically inactive, catalyzing the interconversion ofsubstrate enantiomers, catalyzing the stereochemical inversion aroundthe asymmetric carbon atom in a substrate having only one center ofasymmetry, catalyzing the stereochemical inversion of the configurationaround an asymmetric carbon atom in a substrate having more than oneasymmetric center, and/or catalyzing the racemization of amino acids.

The invention provides isolated, synthetic or recombinant polypeptidescomprising a polypeptide of the invention and lacking a signal sequenceor a prepro sequence. The invention provides isolated, synthetic orrecombinant polypeptides comprising a polypeptide of the invention andhaving a heterologous signal sequence or a heterologous prepro sequence.

In one aspect, a polypeptide of the invention has isomerase activity,e.g., racemase activity, e.g., amino acid racemase activity, alanineracemase activity, and/or epimerase activity comprising a specificactivity at about 37° C. in the range from about 100 to about 1000 unitsper milligram of protein, from about 500 to about 750 units permilligram of protein, from about 500 to about 1200 units per milligramof protein, or from about 750 to about 1000 units per milligram ofprotein. In alternative aspects, polypeptides of the invention haveisomerase activity, e.g., racemase activity, e.g., amino acid racemaseactivity, alanine racemase activity, and/or epimerase activity in therange of between about 0.05 to 20 units per gram, or 0.05, 0.10, 0.20,0.30, 0.40, 0.50, 0.60, 0.70, 0.80, 0.90, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5,4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19 or 20 or more units per gram, where a unit equals one μmol ofproduct released per minute per mg of enzyme. In one embodiment, forracemases, one unit of activity equals one μmol of an isomer withinverted configuration (from the starting isomer) produced per minuteper mg of enzyme (formed from the respective alpha-amino acid or amine).In an alternative embodiment, for amino acid racemases, one unit ofactivity equals one umol of R-amino acid produced per minute per mg ofenzyme formed from the corresponding S-amino acid. In an alternativeembodiment, for amino acid racemases, one unit of activity equals oneumol of S-amino acid produced per minute per mg of enzyme formed fromthe corresponding R-amino acid.

In one aspect, the polypeptides of the invention comprise at least oneglycosylation site or further comprises a polysaccharide. Theglycosylation can be an N-linked glycosylation, e.g., wherein thepolypeptide is glycosylated after being expressed in a P. pastoris or aS. pombe.

The invention provides protein preparation comprising a polypeptide ofthe invention, wherein the protein preparation comprises a liquid, aslurry, a solid or a gel. The invention provides heterodimers comprisinga polypeptide of the invention and a second domain. The second domaincan be a polypeptide and the heterodimer is a fusion protein. the seconddomain can be an epitope or a tag.

The invention provides homodimers or heterodimers comprising apolypeptide of the invention. The invention provides immobilizedpolypeptides, wherein the polypeptide comprises a sequence of theinvention, or a subsequence thereof, or a polypeptide encoded by anucleic acid of the invention, or a polypeptide comprising a polypeptideof the invention and a second domain, e.g., wherein the polypeptide isimmobilized on or inside a cell, a vesicle, a liposome, a film, amembrane, a metal, a resin, a polymer, a ceramic, a glass, amicroelectrode, a graphitic particle, a bead, a gel, a plate, an array,a capillary tube, a crystal, a tablet, a pill, a capsule, a powder, anagglomerate, a surface, a porous structure, or materials such as woodchips, brownstock, pulp, paper, and materials deriving therefrom.

The isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention can be used or formulatedalone or as mixture (a “cocktail”) of isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases, and otherhydrolytic enzymes such as cellulases, mannanases, proteases, lipases,amylases, or redox enzymes such as laccases, peroxidases, catalases,oxidases, or reductases. They can be used formulated in a solid formsuch as a powder, a lyophilized preparation, a granule, a tablet, a bar,a crystal, a capsule, a pill, a pellet, or in a liquid form such as inan aqueous solution, an aerosol, a gel, a paste, a slurry, anaqueous/oil emulsion, a cream, a capsule, or in a vesicular or micellarsuspension. The formulations of the invention can comprise any or acombination of the following ingredients: polyols such as a polyethyleneglycol, a polyvinylalcohol, a glycerol, a sugar such as a sucrose, asorbitol, a trehalose, a glucose, a fructose, a maltose, a mannose, agelling agent such as a guar gum, a carageenan, an alginate, a dextrans,a cellulosic derivative, a pectin, a salt such as a sodium chloride, asodium sulfate, an ammonium sulfate, a calcium chloride, a magnesiumchloride, a zinc chloride, a zinc sulfate, a salt of a fatty acid and afatty acid derivative, a metal chelator such as an EDTA, an EGTA, asodium citrate, an antimicrobial agent such as a fatty acid or a fattyacid derivative, a paraben, a sorbate, a benzoate, an additionalmodulating compound to block the impact of an enzyme such as a protease,a bulk proteins such as a BSA, a wheat hydrolysate, a borate compound,an amino acid or a peptide, an appropriate pH or temperature modulatingcompound, an emulsifier such as a non-ionic and/or an ionic detergent, aredox agent such as a cystine/cysteine, a glutathione, an oxidizedglutathione, a reduced or an antioxidant compound such as an ascorbicacid, or a dispersant. Cross-linking and protein modification such aspegylation, fatty acid modification, glycosylation can also be used toimprove enzyme stability.

The invention provides arrays comprising immobilized polypeptide(s)and/or nucleic acids of the invention, and arrays comprising animmobilized oligonucleotide of the invention. The enzymes, fragmentsthereof and nucleic acids which encode the enzymes, or probes of theinvention, and fragments thereof, can be affixed to a solid support; andthese embodiments can be economical and efficient in the use of enzymesand nucleic acids of the invention in industrial, medical, research,pharmaceutical, food and feed and food and feed supplement processingand other applications and processes. For example, a consortium orcocktail of enzymes (or active fragments thereof), which are used in aspecific chemical reaction, can be attached to a solid support anddunked into a process vat. The enzymatic reaction can occur. Then, thesolid support can be taken out of the vat, along with the enzymesaffixed thereto, for repeated use. In one embodiment of the invention,the isolated nucleic acid is affixed to a solid support. In anotherembodiment of the invention, the solid support is selected from thegroup of a gel, a resin, a polymer, a ceramic, a glass, a microelectrodeand any combination thereof.

For example, solid supports useful in this invention include gels. Someexamples of gels include sepharose, gelatin, glutaraldehyde,chitosan-treated glutaraldehyde, albumin-glutaraldehyde,chitosan-Xanthan, toyopearl gel (polymer gel), alginate,alginate-polylysine, carrageenan, agarose, glyoxyl agarose, magneticagarose, dextran-agarose, poly(Carbamoyl Sulfonate) hydrogel, BSA-PEGhydrogel, phosphorylated polyvinyl alcohol (PVA),monoaminoethyl-N-aminoethyl (MANA), amino, or any combination thereof.Another solid support useful in the present invention are resins orpolymers. Some examples of resins or polymers include cellulose,acrylamide, nylon, rayon, polyester, anion-exchange resin, AMBERLITE™XAD-7, AMBERLITE™ XAD-8, AMBERLITE™ IRA-94, AMBERLITE™ IRC-50,polyvinyl, polyacrylic, polymethacrylate, or any combination thereof.Another type of solid support useful in the present invention isceramic. Some examples include non-porous ceramic, porous ceramic, SiO₂,Al₂O₃. Another type of solid support useful in the present invention isglass. Some examples include non-porous glass, porous glass, aminopropylglass or any combination thereof. Another type of solid support whichcan be used is a microelectrode. An example is apolyethyleneimine-coated magnetite. Graphitic particles can be used as asolid support. Another example of a solid support is a cell, such as ared blood cell.

There are many methods which would be known to one of skill in the artfor immobilizing enzymes or fragments thereof, or nucleic acids, onto asolid support. Some examples of such methods include electrostaticdroplet generation, electrochemical means, via adsorption, via covalentbinding, via cross-linking, via chemical reaction or process, viaencapsulation, via entrapment, via calcium alginate, or via poly(2-hydroxyethyl methacrylate). Like methods are described in Methods inEnzymology, Immobilized Enzymes and Cells, Part C. 1987. Academic Press.Edited by S. P. Colowick and N. O. Kaplan. Volume 136; andImmobilization of Enzymes and Cells. 1997. Humana Press. Edited by G. F.Bickerstaff. Series: Methods in Biotechnology, Edited by J. M. Walker.

The invention provides isolated, synthetic or recombinant antibodiesthat specifically binds to a polypeptide of the invention. The antibodycan be a monoclonal or a polyclonal antibody, or is a single chainedantibody. The invention provides hybridomas comprising an antibody thatspecifically binds to a polypeptide of the invention.

The invention provides methods of isolating or identifying a polypeptidewith an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity comprising the steps of: (a) providing an antibody ofthe invention; (b) providing a sample comprising polypeptides; and (c)contacting the sample of step (b) with the antibody of step (a) underconditions wherein the antibody can specifically bind to thepolypeptide, thereby isolating or identifying a polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity. The invention provides methods of making an anti-isomerase,e.g., anti-racemase, e.g., anti-amino acid racemase, anti-alanineracemase, and/or anti-epimerase antibody comprising administering to anon-human animal a nucleic acid of the invention or a subsequencethereof in an amount sufficient to generate a humoral immune response,thereby making an anti-isomerase, e.g., anti-racemase, e.g., anti-aminoacid racemase, anti-alanine racemase, and/or anti-epimerase antibody.The invention provides methods of making an anti-isomerase, e.g.,anti-racemase, e.g., anti-amino acid racemase, anti-alanine racemase,and/or anti-epimerase antibody comprising administering to a non-humananimal a polypeptide of the invention or a subsequence thereof in anamount sufficient to generate a humoral immune response, thereby makingan anti-isomerase, e.g., anti-racemase, e.g., anti-amino acid racemase,anti-alanine racemase, and/or anti-epimerase antibody.

The invention provides methods of producing a recombinant polypeptidecomprising the steps of: (a) providing a nucleic acid operably linked toa promoter, wherein the nucleic acid comprises a sequence of theinvention; and (b) expressing the nucleic acid of step (a) underconditions that allow expression of the polypeptide, thereby producing arecombinant polypeptide. The method can further comprise transforming ahost cell with the nucleic acid of step (a) followed by expressing thenucleic acid of step (a), thereby producing a recombinant polypeptide ina transformed cell.

The invention provides methods for identifying a polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity comprising: (a) providing a polypeptide of the invention; (b)providing an isomerase, e.g., a racemase, e.g., an amino acid racemase,an alanine racemase, and/or an epimerase substrate; and (c) contactingthe polypeptide with the substrate of step (b) and detecting a decreasein the amount of substrate or an increase in the amount of a reactionproduct, wherein a decrease in the amount of the substrate or anincrease in the amount of the reaction product detects a polypeptidehaving an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity.

The invention provides methods for identifying an isomerase, e.g., aracemase, e.g., an amino acid racemase, an alanine racemase, and/or anepimerase substrate comprising: (a) providing a polypeptide of theinvention; (b) providing a test substrate; and (c) contacting thepolypeptide of step (a) with the test substrate of step (b) anddetecting a decrease in the amount of substrate or an increase in theamount of reaction product, wherein a decrease in the amount of thesubstrate or an increase in the amount of a reaction product identifiesthe test substrate as an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase substrate.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising: (a) expressing a nucleicacid or a vector comprising the nucleic acid under conditions permissivefor translation of the nucleic acid to a polypeptide, wherein thenucleic acid has a sequence of the invention; (b) providing a testcompound; (c) contacting the polypeptide with the test compound; and (d)determining whether the test compound of step (b) specifically binds tothe polypeptide.

The invention provides methods of determining whether a test compoundspecifically binds to a polypeptide comprising: (a) providing apolypeptide of the invention; (b) providing a test compound; (c)contacting the polypeptide with the test compound; and (d) determiningwhether the test compound of step (b) specifically binds to thepolypeptide.

The invention provides methods for identifying a modulator of anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity comprising: (a) providing a polypeptide of the invention; (b)providing a test compound; (c) contacting the polypeptide of step (a)with the test compound of step (b) and measuring an activity of theisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase, wherein a change in the isomerase activity, e.g.,racemase activity, e.g., amino acid racemase activity, alanine racemaseactivity, and/or epimerase activity measured in the presence of the testcompound compared to the activity in the absence of the test compoundprovides a determination that the test compound modulates the isomeraseactivity, e.g., racemase activity, e.g., amino acid racemase activity,alanine racemase activity, and/or epimerase activity. The isomeraseactivity, e.g., racemase activity, e.g., amino acid racemase activity,alanine racemase activity, and/or epimerase activity can be measured byproviding an isomerase, e.g., a racemase, e.g., an amino acid racemase,an alanine racemase, and/or an epimerase substrate and detecting adecrease in the amount of the substrate or an increase in the amount ofa reaction product, or, an increase in the amount of the substrate or adecrease in the amount of a reaction product. In one aspect, a decreasein the amount of the substrate or an increase in the amount of thereaction product with the test compound as compared to the amount ofsubstrate or reaction product without the test compound identifies thetest compound as an activator of an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity. In one aspect, an increase inthe amount of the substrate or a decrease in the amount of the reactionproduct with the test compound as compared to the amount of substrate orreaction product without the test compound identifies the test compoundas an inhibitor of an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity.

The invention provides computer systems comprising a processor and adata storage device wherein said data storage device has stored thereona polypeptide sequence or a nucleic acid sequence, wherein thepolypeptide sequence comprises sequence of the invention, a polypeptideencoded by a nucleic acid of the invention. The computer systems canfurther comprise a sequence comparison algorithm and a data storagedevice having at least one reference sequence stored thereon. In anotheraspect, the sequence comparison algorithm comprises a computer programthat indicates polymorphisms. In one aspect, the computer system canfurther comprise an identifier that identifies one or more features insaid sequence. The invention provides computer readable media havingstored thereon a polypeptide sequence or a nucleic acid sequence of theinvention. The invention provides methods for identifying a feature in asequence comprising the steps of: (a) reading the sequence using acomputer program which identifies one or more features in a sequence,wherein the sequence comprises a polypeptide sequence or a nucleic acidsequence of the invention; and (b) identifying one or more features inthe sequence with the computer program. The invention provides methodsfor comparing a first sequence to a second sequence comprising the stepsof: (a) reading the first sequence and the second sequence through useof a computer program which compares sequences, wherein the firstsequence comprises a polypeptide sequence or a nucleic acid sequence ofthe invention; and (b) determining differences between the firstsequence and the second sequence with the computer program. The step ofdetermining differences between the first sequence and the secondsequence can further comprise the step of identifying polymorphisms. Inone aspect, the method can further comprise an identifier thatidentifies one or more features in a sequence. In another aspect, themethod can comprise reading the first sequence using a computer programand identifying one or more features in the sequence.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity from an environmentalsample comprising the steps of: (a) providing an amplification primersequence pair for amplifying a nucleic acid encoding a polypeptidehaving an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity, wherein the primer pair is capable of amplifying anucleic acid of the invention; (b) isolating a nucleic acid from theenvironmental sample or treating the environmental sample such thatnucleic acid in the sample is accessible for hybridization to theamplification primer pair; and, (c) combining the nucleic acid of step(b) with the amplification primer pair of step (a) and amplifyingnucleic acid from the environmental sample, thereby isolating orrecovering a nucleic acid encoding a polypeptide having an isomeraseactivity, e.g., a racemase activity, e.g., an amino acid racemaseactivity, an alanine racemase activity, and/or an epimerase activityfrom an environmental sample. One or each member of the amplificationprimer sequence pair can comprise an oligonucleotide comprising at leastabout 10 to 50 consecutive bases of a sequence of the invention. In oneaspect, the amplification primer sequence pair is an amplification pairof the invention.

The invention provides methods for isolating or recovering a nucleicacid encoding a polypeptide having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity from an environmentalsample comprising the steps of: (a) providing a polynucleotide probecomprising a nucleic acid of the invention or a subsequence thereof; (b)isolating a nucleic acid from the environmental sample or treating theenvironmental sample such that nucleic acid in the sample is accessiblefor hybridization to a polynucleotide probe of step (a); (c) combiningthe isolated nucleic acid or the treated environmental sample of step(b) with the polynucleotide probe of step (a); and (d) isolating anucleic acid that specifically hybridizes with the polynucleotide probeof step (a), thereby isolating or recovering a nucleic acid encoding apolypeptide having an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity from an environmental sample. Theenvironmental sample can comprise a water sample, a liquid sample, asoil sample, an air sample or a biological sample. In one aspect, thebiological sample can be derived from a bacterial cell, a protozoancell, an insect cell, a yeast cell, a plant cell, a fungal cell or amammalian cell.

The invention provides methods of generating a variant of a nucleic acidencoding a polypeptide having an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity comprising the steps of: (a)providing a template nucleic acid comprising a nucleic acid of theinvention; and (b) modifying, deleting or adding one or more nucleotidesin the template sequence, or a combination thereof, to generate avariant of the template nucleic acid. In one aspect, the method canfurther comprise expressing the variant nucleic acid to generate avariant isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase polypeptide. The modifications, additions ordeletions can be introduced by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (e.g., GeneReassembly, see, e.g., U.S. Pat.No. 6,537,776), Gene Site Saturation Mutagenesis (GSSM), syntheticligation reassembly (SLR) or a combination thereof. In another aspect,the modifications, additions or deletions are introduced by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

In one aspect, the method can be iteratively repeated until anisomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase having an altered or different activity oran altered or different stability from that of a polypeptide encoded bythe template nucleic acid is produced. In one aspect, the variantisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase polypeptide is thermotolerant, and retains someactivity after being exposed to an elevated temperature. In anotheraspect, the variant isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase polypeptide has increasedglycosylation as compared to the isomerase, e.g., the racemase, e.g.,the amino acid racemase, the alanine racemase, and/or the epimeraseencoded by a template nucleic acid. Alternatively, the variantisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase polypeptide has an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity under a high temperature, whereinthe isomerase, e.g., the racemase, e.g., the amino acid racemase, thealanine racemase, and/or the epimerase encoded by the template nucleicacid is not active under the high temperature. In one aspect, the methodcan be iteratively repeated until an isomerase, e.g., a racemase, e.g.,an amino acid racemase, an alanine racemase, and/or an epimerase codingsequence having an altered codon usage from that of the template nucleicacid is produced. In another aspect, the method can be iterativelyrepeated until an isomerase, e.g., a racemase, e.g., an amino acidracemase, an alanine racemase, and/or an epimerase gene having higher orlower level of message expression or stability from that of the templatenucleic acid is produced. In another aspect, formulation of the finalisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase product enables an increase or modulation of theperformance of the isomerase, e.g., the racemase, e.g., the amino acidracemase, the alanine racemase, and/or the epimerase in the product.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity to increase its expression in ahost cell, the method comprising: (a) providing a nucleic acid of theinvention encoding a polypeptide having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity; and, (b) identifying anon-preferred or a less preferred codon in the nucleic acid of step (a)and replacing it with a preferred or neutrally used codon encoding thesame amino acid as the replaced codon, wherein a preferred codon is acodon over-represented in coding sequences in genes in the host cell anda non-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell, thereby modifying thenucleic acid to increase its expression in a host cell.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity; the method comprising: (a)providing a nucleic acid of the invention; and, (b) identifying a codonin the nucleic acid of step (a) and replacing it with a different codonencoding the same amino acid as the replaced codon, thereby modifyingcodons in a nucleic acid encoding an isomerase, e.g., a racemase, e.g.,an amino acid racemase, an alanine racemase, and/or an epimerase.

The invention provides methods for modifying codons in a nucleic acidencoding a polypeptide having an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity to increase its expression in ahost cell, the method comprising: (a) providing a nucleic acid of theinvention encoding an isomerase, e.g., a racemase, e.g., an amino acidracemase, an alanine racemase, and/or an epimerase polypeptide; and, (b)identifying a non-preferred or a less preferred codon in the nucleicacid of step (a) and replacing it with a preferred or neutrally usedcodon encoding the same amino acid as the replaced codon, wherein apreferred codon is a codon over-represented in coding sequences in genesin the host cell and a non-preferred or less preferred codon is a codonunder-represented in coding sequences in genes in the host cell, therebymodifying the nucleic acid to increase its expression in a host cell.

The invention provides methods for modifying a codon in a nucleic acidencoding a polypeptide having an isomerase activity, e.g., a racemaseactivity, e.g., an amino acid racemase activity, an alanine racemaseactivity, and/or an epimerase activity to decrease its expression in ahost cell, the method comprising: (a) providing a nucleic acid of theinvention; and (b) identifying at least one preferred codon in thenucleic acid of step (a) and replacing it with a non-preferred or lesspreferred codon encoding the same amino acid as the replaced codon,wherein a preferred codon is a codon over-represented in codingsequences in genes in a host cell and a non-preferred or less preferredcodon is a codon under-represented in coding sequences in genes in thehost cell, thereby modifying the nucleic acid to decrease its expressionin a host cell. In one aspect, the host cell can be a bacterial cell, afungal cell, an insect cell, a yeast cell, a plant cell or a mammaliancell.

The invention provides methods for producing a library of nucleic acidsencoding a plurality of modified isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase active sites orsubstrate binding sites, wherein the modified active sites or substratebinding sites are derived from a first nucleic acid comprising asequence encoding a first active site or a first substrate binding sitethe method comprising: (a) providing a first nucleic acid encoding afirst active site or first substrate binding site, wherein the firstnucleic acid sequence comprises a sequence that hybridizes understringent conditions to a sequence of the invention, or a subsequencethereof, and the nucleic acid encodes an isomerase, e.g., a racemase,e.g., an amino acid racemase, an alanine racemase, and/or an epimeraseactive site or an isomerase, e.g., a racemase, e.g., an amino acidracemase, an alanine racemase, and/or an epimerase substrate bindingsite; (b) providing a set of mutagenic oligonucleotides that encodenaturally-occurring amino acid variants at a plurality of targetedcodons in the first nucleic acid; and, (c) using the set of mutagenicoligonucleotides to generate a set of active site-encoding or substratebinding site-encoding variant nucleic acids encoding a range of aminoacid variations at each amino acid codon that was mutagenized, therebyproducing a library of nucleic acids encoding a plurality of modifiedisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase active sites or substrate binding sites. In one aspect,the method comprises mutagenizing the first nucleic acid of step (a) bya method comprising an optimized directed evolution system, Gene SiteSaturation Mutagenesis (GSSM), or a synthetic ligation reassembly (SLR).In one aspect, the method comprises mutagenizing the first nucleic acidof step (a) or variants by a method comprising error-prone PCR,shuffling, oligonucleotide-directed mutagenesis, assembly PCR, sexualPCR mutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (GeneReassembly, U.S. Pat. No. 6,537,776),Gene Site Saturation Mutagenesis (GSSM), synthetic ligation reassembly(SLR) and a combination thereof. In one aspect, the method comprisesmutagenizing the first nucleic acid of step (a) or variants by a methodcomprising recombination, recursive sequence recombination,phosphothioate-modified DNA mutagenesis, uracil-containing templatemutagenesis, gapped duplex mutagenesis, point mismatch repairmutagenesis, repair-deficient host strain mutagenesis, chemicalmutagenesis, radiogenic mutagenesis, deletion mutagenesis,restriction-selection mutagenesis, restriction-purification mutagenesis,artificial gene synthesis, ensemble mutagenesis, chimeric nucleic acidmultimer creation and a combination thereof.

The invention provides methods for making a small molecule comprising:(a) providing a plurality of biosynthetic enzymes capable ofsynthesizing or modifying a small molecule, wherein one of the enzymescomprises an isomerase, e.g., a racemase, e.g., an amino acid racemase,an alanine racemase, and/or an epimerase enzyme encoded by a nucleicacid of the invention; (b) providing a substrate for at least one of theenzymes of step (a); and (c) reacting the substrate of step (b) with theenzymes under conditions that facilitate a plurality of biocatalyticreactions to generate a small molecule by a series of biocatalyticreactions. The invention provides methods for modifying a small moleculecomprising: (a) providing an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase enzyme, whereinthe enzyme comprises a polypeptide of the invention, or, a polypeptideencoded by a nucleic acid of the invention, or a subsequence thereof;(b) providing a small molecule; and (c) reacting the enzyme of step (a)with the small molecule of step (b) under conditions that facilitate anenzymatic reaction catalyzed by the isomerase, e.g., the racemase, e.g.,the amino acid racemase, the alanine racemase, and/or the epimeraseenzyme, thereby modifying a small molecule by an isomerase, e.g., aracemase, e.g., an amino acid racemase, an alanine racemase, and/or anepimerase enzymatic reaction. In one aspect, the method can comprise aplurality of small molecule substrates for the enzyme of step (a),thereby generating a library of modified small molecules produced by atleast one enzymatic reaction catalyzed by the isomerase, e.g., theracemase, e.g., the amino acid racemase, the alanine racemase, and/orthe epimerase enzyme. In one aspect, the method can comprise a pluralityof additional enzymes under conditions that facilitate a plurality ofbiocatalytic reactions by the enzymes to form a library of modifiedsmall molecules produced by the plurality of enzymatic reactions. Inanother aspect, the method can further comprise the step of testing thelibrary to determine if a particular modified small molecule thatexhibits a desired activity is present within the library. The step oftesting the library can further comprise the steps of systematicallyeliminating all but one of the biocatalytic reactions used to produce aportion of the plurality of the modified small molecules within thelibrary by testing the portion of the modified small molecule for thepresence or absence of the particular modified small molecule with adesired activity, and identifying at least one specific biocatalyticreaction that produces the particular modified small molecule of desiredactivity.

The invention provides methods for determining a functional fragment ofan isomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase enzyme comprising the steps of: (a)providing an isomerase, e.g., a racemase, e.g., an amino acid racemase,an alanine racemase, and/or an epimerase enzyme, wherein the enzymecomprises a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention, or a subsequence thereof; and (b)deleting a plurality of amino acid residues from the sequence of step(a) and testing the remaining subsequence for an isomerase activity,e.g., a racemase activity, e.g., an amino acid racemase activity, analanine racemase activity, and/or an epimerase activity, therebydetermining a functional fragment of an isomerase, e.g., a racemase,e.g., an amino acid racemase, an alanine racemase, and/or an epimeraseenzyme. In one aspect, the isomerase activity, e.g., racemase activity,e.g., amino acid racemase activity, alanine racemase activity, and/orepimerase activity is measured by providing an isomerase, e.g., aracemase, e.g., an amino acid racemase, an alanine racemase, and/or anepimerase substrate and detecting a decrease in the amount of thesubstrate or an increase in the amount of a reaction product.

The invention provides methods for whole cell engineering of new ormodified phenotypes by using real-time metabolic flux analysis, themethod comprising: (a) making a modified cell by modifying the geneticcomposition of a cell, wherein the genetic composition is modified byaddition to the cell of a nucleic acid of the invention; (b) culturingthe modified cell to generate a plurality of modified cells; (c)measuring at least one metabolic parameter of the cell by monitoring thecell culture of step (b) in real time; and, (d) analyzing the data ofstep (c) to determine if the measured parameter differs from acomparable measurement in an unmodified cell under similar conditions,thereby identifying an engineered phenotype in the cell using real-timemetabolic flux analysis. In one aspect, the genetic composition of thecell can be modified by a method comprising deletion of a sequence ormodification of a sequence in the cell, or, knocking out the expressionof a gene. In one aspect, the method can further comprise selecting acell comprising a newly engineered phenotype. In another aspect, themethod can comprise culturing the selected cell, thereby generating anew cell strain comprising a newly engineered phenotype.

The invention provides isolated, synthetic or recombinant signalsequences consisting of, or comprising, a sequence as set forth inresidues 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18,1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26,1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34,1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 40, 1 to 41, 1 to 42, 1 to 43or 1 to 44, of a polypeptide of the invention, including exemplarypolypeptide sequences of the invention.

The invention provides chimeric polypeptides comprising at least a firstdomain comprising a signal peptide (SP) and at least a second domaincomprising a heterologous polypeptide or peptide comprising a sequenceof the invention, or a subsequence thereof, wherein the heterologouspolypeptide or peptide is not naturally associated with the signalpeptide (SP). In one aspect, the signal peptide (SP) is not derived froman isomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase. The heterologous polypeptide or peptidecan be amino terminal to, carboxy terminal to or on both ends of thesignal peptide (SP) or an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase catalytic domain(CD). The invention provides isolated, synthetic or recombinant nucleicacids encoding a chimeric polypeptide, wherein the chimeric polypeptidecomprises at least a first domain comprising signal peptide (SP) and atleast a second domain comprising a heterologous polypeptide or peptidecomprising a sequence of the invention, or a subsequence thereof,wherein the heterologous polypeptide or peptide is not naturallyassociated with the signal peptide (SP).

The invention provides methods of increasing thermotolerance orthermostability of an isomerase, e.g., a racemase, e.g., an amino acidracemase, an alanine racemase, and/or an epimerase polypeptide, themethod comprising glycosylating an isomerase, e.g., a racemase, e.g., anamino acid racemase, an alanine racemase, and/or an epimerasepolypeptide, wherein the polypeptide comprises at least thirtycontiguous amino acids of a polypeptide of the invention; or apolypeptide encoded by a nucleic acid sequence of the invention, therebyincreasing the thermotolerance or thermostability of the isomerase,e.g., the racemase, e.g., the amino acid racemase, the alanine racemase,and/or the epimerase polypeptide. In one aspect, the isomerase, e.g.,the racemase, e.g., the amino acid racemase, the alanine racemase,and/or the epimerase-specific activity can be thermostable orthermotolerant at a temperature in the range from greater than about 0°C. to about 20° C., about 20° C. to about 37° C., about 37° C. to about50° C., about 50° C. to about 70° C., about 70° C. to about 75° C.,about 75° C. to about 80° C., about 80° C. to about 85° C., about 85° C.to about 90° C., about 90° C. to about 95° C., about 95° C. to about100° C., about 100° C. to about 110° C., or higher.

The invention provides methods for overexpressing a recombinantisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase polypeptide in a cell comprising expressing a vectorcomprising a nucleic acid comprising a nucleic acid of the invention ora nucleic acid sequence of the invention, wherein the sequenceidentities are determined by analysis with a sequence comparisonalgorithm or by visual inspection, wherein overexpression is effected byuse of a high activity promoter, a dicistronic vector or by geneamplification of the vector.

The invention provides methods of making a transgenic plant and seedscomprising: (a) introducing a heterologous nucleic acid sequence intothe cell, wherein the heterologous nucleic sequence comprises a nucleicacid sequence of the invention, thereby producing a transformed plant orseed cell; and (b) producing a transgenic plant from the transformedcell or seed. In one aspect, the step (a) can further compriseintroducing the heterologous nucleic acid sequence by electroporation ormicroinjection of plant cell protoplasts. In another aspect, the step(a) can further comprise introducing the heterologous nucleic acidsequence directly to plant tissue by DNA particle bombardment.Alternatively, the step (a) can further comprise introducing theheterologous nucleic acid sequence into the plant cell DNA using anAgrobacterium tumefaciens host. In one aspect, the plant cell can be apotato, corn, rice, wheat, tobacco, or barley cell.

The invention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising: (a) transforming the plant cellwith a heterologous nucleic acid sequence operably linked to a promoter,wherein the heterologous nucleic sequence comprises a nucleic acid ofthe invention; (b) growing the plant under conditions wherein theheterologous nucleic acids sequence is expressed in the plant cell. Theinvention provides methods of expressing a heterologous nucleic acidsequence in a plant cell comprising: (a) transforming the plant cellwith a heterologous nucleic acid sequence operably linked to a promoter,wherein the heterologous nucleic sequence comprises a sequence of theinvention; (b) growing the plant under conditions wherein theheterologous nucleic acids sequence is expressed in the plant cell.

The invention provides detergent compositions comprising a polypeptideof the invention, or a polypeptide encoded by a nucleic acid of theinvention, wherein the polypeptide has an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity. The isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasecan be nonsurface-active or surface-active. The isomerase, e.g., theracemase, e.g., the amino acid racemase, the alanine racemase, and/orthe epimerase can be formulated in a non-aqueous liquid composition, acast solid, a granular form, a particulate form, a compressed tablet, agel form, a paste or a slurry form. The invention provides methods forwashing an object comprising: (a) providing a composition comprising apolypeptide of the invention having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity, or a polypeptideencoded by a nucleic acid of the invention; (b) providing an object; and(c) contacting the polypeptide of step (a) and the object of step (b)under conditions wherein the composition can wash the object.

The invention provides textiles or fabrics, including, e.g., threads,comprising a polypeptide of the invention, or a polypeptide encoded by anucleic acid of the invention. The invention provides methods fortreating a textile or fabric (e.g., removing a stain from a composition)comprising: (a) providing a composition comprising a polypeptide of theinvention having an isomerase activity, e.g., a racemase activity, e.g.,an amino acid racemase activity, an alanine racemase activity, and/or anepimerase activity, or a polypeptide encoded by a nucleic acid of theinvention; (b) providing a textile or fabric; and (c) contacting thepolypeptide of step (a) and the composition of step (b) under conditionswherein the isomerase, e.g., the racemase, e.g., the amino acidracemase, the alanine racemase, and/or the epimerase can treat thetextile or fabric (e.g., remove the stain). The invention providesmethods for improving the finish of a fabric comprising: (a) providing acomposition comprising a polypeptide of the invention having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, or a polypeptide encoded by a nucleic acid of the invention;(b) providing a fabric; and (c) contacting the polypeptide of step (a)and the fabric of step (b) under conditions wherein the polypeptide cantreat the fabric thereby improving the finish of the fabric. In oneaspect, the fabric is a wool or a silk. In another aspect, the fabric isa cellulosic fiber or a blend of a natural fiber and a synthetic fiber.

The invention provides feeds, foods, feed supplements, food supplements,dietary compositions or dietary aids comprising a polypeptide of theinvention, or a polypeptide encoded by a nucleic acid of the invention.The food or the feed can be, e.g., a cereal, a grain, a corn and thelike.

The invention provides dough, bread or baked products and/or dough,bread or baked product precursors comprising a polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, wherein the polypeptide comprises a sequence of the invention,or the polypeptide is encoded by a nucleic acid comprising a sequence ofthe invention, or an enzymatically active fragment thereof.

The invention provides beverages and beverage precursors comprising apolypeptide, or an enzymatically active fragment thereof, having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, wherein the polypeptide comprises a sequence of the invention,or the polypeptide is encoded by a nucleic acid comprising a sequence ofthe invention. The invention provides methods of beverage productioncomprising administration of at least one polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, wherein the polypeptide comprises a sequence of the invention,or the polypeptide is encoded by a nucleic acid comprising a sequence ofthe invention, or an enzymatically active fragment thereof, to abeverage or a beverage precursor, wherein in one aspect (optionally) thebeverage or beverage precursor is a wort or a beer.

The invention provides food, feed or nutritional supplements, e.g. for ahuman or an animal comprising a polypeptide of the invention, e.g., apolypeptide encoded by the nucleic acid of the invention. In one aspect,the polypeptide in the food or nutritional supplement can beglycosylated. The invention provides edible enzyme delivery matricescomprising a polypeptide of the invention, e.g., a polypeptide encodedby the nucleic acid of the invention. In one aspect, the delivery matrixcomprises a pellet. In one aspect, the polypeptide can be glycosylated.In one aspect, the isomerase activity, e.g., racemase activity, e.g.,amino acid racemase activity, alanine racemase activity, and/orepimerase activity is thermotolerant. In another aspect, the isomeraseactivity, e.g., racemase activity, e.g., amino acid racemase activity,alanine racemase activity, and/or epimerase activity is thermostable.

In one aspect, the isomerase, e.g., the racemase, e.g., the amino acidracemase, the alanine racemase, and/or the epimerase enzyme can beprepared by expression of a polynucleotide encoding the isomerase, e.g.,the racemase, e.g., the amino acid racemase, the alanine racemase,and/or the epimerase in an organism selected from the group consistingof a bacterium, a yeast, a plant, an insect, a fungus and an animal. Theorganism can be selected from the group consisting of an S. pombe, S.cerevisiae, Pichia pastoris, Pseudomonas sp., E. coli, Streptomyces sp.,Bacillus sp. and Lactobacillus sp.

The invention provides edible enzyme delivery matrix comprising athermostable recombinant isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase enzyme, e.g., a polypeptideof the invention. The invention provides methods for delivering anisomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase supplement to an animal, the methodcomprising: preparing an edible enzyme delivery matrix in the form ofpellets comprising a granulate edible carrier and a thermostablerecombinant isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase enzyme, wherein the pellets readilydisperse the isomerase, e.g., the racemase, e.g., the amino acidracemase, the alanine racemase, and/or the epimerase enzyme containedtherein into aqueous media, and administering the edible enzyme deliverymatrix to the animal. The recombinant isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase enzyme cancomprise a polypeptide of the invention. The granulate edible carriercan comprise a carrier selected from the group consisting of a graingerm, a grain germ that is spent of oil, a hay, an alfalfa, a timothy, asoy hull, a sunflower seed meal and a wheat midd. The edible carrier cancomprise grain germ that is spent of oil. The isomerase, e.g., theracemase, e.g., the amino acid racemase, the alanine racemase, and/orthe epimerase enzyme can be glycosylated to provide thermostability atpelletizing conditions. The delivery matrix can be formed by pelletizinga mixture comprising a grain germ and an isomerase, e.g., a racemase,e.g., an amino acid racemase, an alanine racemase, and/or an epimerase.The pelletizing conditions can include application of steam. Thepelletizing conditions can comprise application of a temperature inexcess of about 80° C. for about 5 minutes and the enzyme retains aspecific activity of at least 350 to about 900 units per milligram ofenzyme.

The invention provides methods for treating, e.g. improving texture andflavor of a dairy product comprising: (a) providing a polypeptide of theinvention having an isomerase activity, e.g., a racemase activity, e.g.,an amino acid racemase activity, an alanine racemase activity, and/or anepimerase activity, or an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase encoded by anucleic acid of the invention; (b) providing a dairy product; and (c)contacting the polypeptide of step (a) and the dairy product of step (b)under conditions wherein the isomerase, e.g., the racemase, e.g., theamino acid racemase, the alanine racemase, and/or the epimerase cantreat, e.g. improve the texture or flavor of the dairy product. In oneaspect, the dairy product comprises a cheese or a yogurt. The inventionprovides dairy products comprising an isomerase, e.g., a racemase, e.g.,an amino acid racemase, an alanine racemase, and/or an epimerase of theinvention, or is encoded by a nucleic acid of the invention.

The invention provides methods for improving the extraction of oil froman oil-rich plant material comprising: (a) providing a polypeptide ofthe invention having an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity, or an isomerase, e.g., a racemase, e.g.,an amino acid racemase, an alanine racemase, and/or an epimerase encodedby a nucleic acid of the invention; (b) providing an oil-rich plantmaterial; and (c) contacting the polypeptide of step (a) and theoil-rich plant material. In one aspect, the oil-rich plant materialcomprises an oil-rich seed. The oil can be a soybean oil, an olive oil,a rapeseed (canola) oil or a sunflower oil.

The invention provides methods for preparing a fruit or vegetable juice,syrup, puree or extract comprising: (a) providing a polypeptide of theinvention having an isomerase activity, e.g., a racemase activity, e.g.,an amino acid racemase activity, an alanine racemase activity, and/or anepimerase activity, or an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase encoded by anucleic acid of the invention; (b) providing a composition or a liquidcomprising a fruit or vegetable material; and (c) contacting thepolypeptide of step (a) and the composition, thereby preparing the fruitor vegetable juice, syrup, puree or extract.

The invention provides methods for treating a wood, a wood product, apaper, a paper product, a pulp, a pulp product, a paper waste or a paperrecycling composition comprising: (a) providing a polypeptide of theinvention having an isomerase activity, e.g., a racemase activity, e.g.,an amino acid racemase activity, an alanine racemase activity, and/or anepimerase activity, or an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase encoded by anucleic acid of the invention; (b) providing a composition comprising awood, a wood product, a paper, a paper product, a pulp, a pulp product,a paper waste or a paper recycling composition; and (c) contacting thepolypeptide of step (a) and the composition, thereby treating the wood,wood product, paper, paper product, pulp, pulp product, paper waste orpaper recycling composition. In one aspect of the invention, thetreatment comprises reducing or solubilizing lignin (delignification),bleaching or decoloring, and/or deinking.

The invention provides papers or paper products or paper pulp comprisingan isomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase of the invention, or a polypeptide encodedby a nucleic acid of the invention. The invention provides methods fortreating a paper or a paper or wood pulp comprising: (a) providing apolypeptide of the invention having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity, or an isomerase, e.g.,a racemase, e.g., an amino acid racemase, an alanine racemase, and/or anepimerase encoded by a nucleic acid of the invention; (b) providing acomposition comprising a paper or a paper or wood pulp; and (c)contacting the polypeptide of step (a) and the composition of step (b)under conditions wherein the isomerase, e.g., the racemase, e.g., theamino acid racemase, the alanine racemase, and/or the epimerase cantreat the paper or paper or wood pulp.

The invention provides methods for bleaching a thread, fabric, yarn,cloth or textile comprising contacting the fabric, yarn, cloth ortextile with an isomerase, e.g., a racemase, e.g., an amino acidracemase, an alanine racemase, and/or an epimerase under conditionssuitable to produce a whitening of the textile, wherein the isomerase,e.g., the racemase, e.g., the amino acid racemase, the alanine racemase,and/or the epimerase comprises a polypeptide of the invention, or anenzymatically active fragment thereof. The thread, fabric, yarn, clothor textile can comprise a non-cotton cellulosic thread, fabric, yarn,cloth or textile. The invention provides fabrics, yarns, cloths ortextiles comprising a polypeptide having a sequence of the invention, ora polypeptide encoded by a nucleic acid comprising a sequence of theinvention, or an enzymatically active fragment thereof, wherein in oneaspect (optionally) the fabric, yarn, cloth or textile comprises anon-cotton cellulosic fabric, yarn, cloth or textile.

The invention provides wood, wood chips, wood pulp, wood products, paperpulps, paper products, newspapers or paper waste comprising apolypeptide of the invention, or an enzymatically active fragmentthereof. The invention provides thread, fabric, yarn, cloth or textilecomprising a polypeptide of the invention, or an enzymatically activefragment thereof.

The invention provides methods for making ethanol comprising contactingan organic material, e.g. a biomass with a polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, wherein the polypeptide has a sequence of the invention, orthe polypeptide is encoded by a nucleic acid comprising a sequence ofthe invention, or an enzymatically active fragment thereof. Theinvention provides compositions comprising an ethanol and a polypeptidehaving an isomerase activity, e.g., a racemase activity, e.g., an aminoacid racemase activity, an alanine racemase activity, and/or anepimerase activity, wherein the polypeptide has a sequence of theinvention, or the polypeptide is encoded by a nucleic acid comprising asequence of the invention, or an enzymatically active fragment thereof.The invention provides methods for making ethanol comprising: (a)providing at least one polypeptide having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity, or an enzymaticallyactive fragment thereof; (b) providing an organic composition; and (c)contacting the composition of step (b) with the polypeptide of step (a).

The invention provides methods of making a pharmaceutical (drug)composition, a pharmaceutical (drug) precursor, or a drug intermediate,comprising using a polypeptide of this invention having an isomeraseactivity, e.g., a racemase activity, e.g., an amino acid racemaseactivity, an alanine racemase activity, and/or an epimerase activity.

The invention provides pharmaceutical (drug) compositions andpharmaceutical (drug) precursors and intermediates comprising apolypeptide having an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity, wherein the polypeptide comprises asequence of the invention, or the polypeptide is encoded by a nucleicacid comprising a sequence of the invention, or an enzymatically activefragment thereof. In one aspect, the pharmaceutical composition acts asa digestive aid, is an antibiotic or is useful for treatment of aminoacid deficiencies. In one aspect, the treatment is prophylactic.

In one aspect, the invention provides oral care products comprising apolypeptide of the invention having an isomerase activity, e.g., aracemase activity, e.g., an amino acid racemase activity, an alanineracemase activity, and/or an epimerase activity, or an isomerase, e.g.,a racemase, e.g., an amino acid racemase, an alanine racemase, and/or anepimerase encoded by a nucleic acid of the invention. The oral careproduct can comprise a toothpaste, a dental cream, a gel or a toothpowder, an odontic, a mouth wash, a pre- or post brushing rinseformulation, a chewing gum, a lozenge or a candy. The invention providescontact lens cleaning compositions comprising a polypeptide of theinvention having an isomerase activity, e.g., a racemase activity, e.g.,an amino acid racemase activity, an alanine racemase activity, and/or anepimerase activity, or an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase encoded by anucleic acid of the invention.

The invention provides chimeric isomerases, e.g., racemases, e.g., aminoacid racemases, alanine racemases, and/or epimerases comprising apolypeptide sequence of the invention and at least one heterologousdomain, e.g. a binding domain or a dockerin domain. The inventionprovides methods for designing a chimeric isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase having anew specificity or an enhanced specificity, comprising inserting aheterologous or an additional endogenous domain, e.g. a binding domainor a dockerin domain, into an isomerase, e.g., a racemase, e.g., anamino acid racemase, an alanine racemase, and/or an epimerase, whereinthe domain is inserted into an isomerase, e.g., a racemase, e.g., anamino acid racemase, an alanine racemase, and/or an epimerase sequenceof the invention.

The invention provides enzyme mixtures, or “cocktails” comprising atleast one enzyme of the invention and one or more other enzyme(s), whichcan be another isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase, or any other enzyme; for example,the “cocktails” of the invention, in addition to at least one enzyme ofthis invention, can comprise any other enzyme, such as xylanase,cellulases, lipases, esterases, proteases, or endoglycosidases,endo-beta.-1,4-glucanases, beta-glucanases, endo-beta-1,3(4)-glucanases,cutinases, peroxidases, catalases, laccases, amylases, glucoamylases,pectinases, transferases, transaminases, amino transferases,dehydrogenases, oxidoreductases, reductases, oxidases, phenoloxidases,ligninases, pullulanases, arabinanases, hemicellulases, mannanases,xyloglucanases, pectin acetyl esterases, rhamnogalacturonan acetylesterases, polygalacturonases, rhamnogalacturonases, galactanases,pectin lyases, pectin methylesterases, cellobiohydrolases and/ortransglutaminases, to name just a few examples. In alternativeembodiments, these enzyme mixtures, or “cocktails” comprising at leastone enzyme of the invention can be used in any process or method of theinvention, or composition of the invention, e.g., in foods or feeds,food or feed supplements, textiles, papers, processed woods, etc. andmethods for making them, and in compositions and methods for treatingpaper, pulp, wood, paper, pulp or wood waste or by-products, and thelike, and in the final products thereof.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

All publications, patents, patent applications, GenBank sequences andATCC deposits, cited herein are hereby expressly incorporated byreference for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings are illustrative of aspects of the invention andare not meant to limit the scope of the invention as encompassed by theclaims.

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is a block diagram of a computer system.

FIG. 2 is a flow diagram illustrating one aspect of a process forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database.

FIG. 3 is a flow diagram illustrating one aspect of a process in acomputer for determining whether two sequences are homologous.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence.

Like reference symbols in the various drawings indicate like elements.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases, and polynucleotidesencoding them and methods of making and using them. Isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases, of the polypeptides of the invention encompasses enzymeshaving isomerase activity, e.g., racemase activity, e.g., amino acidracemase activity, alanine racemase activity, and/or epimerase activity,and/or catalyze the re-arrangement of atoms within a molecule, catalyzethe conversion of one isomer into another, catalyze the conversion of anoptically active substrate into a raceme, which is optically inactive,catalyze the interconversion of substrate enantiomers, catalyze thestereochemical inversion around the asymmetric carbon atom in asubstrate having only one center of asymmetry, catalyze thestereochemical inversion of the configuration around an asymmetriccarbon atom in a substrate having more than one asymmetric center,and/or catalyze the racemization of amino acids. The enzymes, e.g.,racemases of the invention can be used to make and/or processpharmaceutical (drug) compositions, pharmaceutical (drug) precursors andintermediates, such as molecules comprising unnatural amino acids orantibiotics, sweeteners, peptide enzymes, peptide hormones, fuel andfuel additive compositions, foods and food additives, beverage andbeverage additives, feeds and feed additives, drugs and drug additives,dietary supplements, textiles, wood, paper, pulp, detergents and thelike.

In one aspect, an enzyme of the invention is thermotolerant and/ortolerant of high and/or low pH conditions. For example, in one aspect,an isomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase of the invention retains activity underconditions comprising a temperature of at least about 80° C., 85° C.,86° C., 87° C., 88° C., 89° C., 90° C., 91° C., 92° C., 93° C., 94° C.,95° C., 96° C., 97° C., 98° C., 99° C., 100° C., 101° C., 102° C., 103°C., 103.5° C., 104° C., 105° C., 107° C., 108° C., 109° C. or 110° C.,or more, and a basic pH of at least about pH 11, or more.

The invention provides isolated, synthetic or recombinant nucleic acidscomprising a nucleic acid encoding at least one polypeptide having anisomerase activity, e.g., a racemase activity, e.g., an amino acidracemase activity, an alanine racemase activity, and/or an epimeraseactivity, or other activity as described herein, wherein the nucleicacid comprises a sequence having at least about 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or more, or complete (100%) sequence identity (homology)to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ IDNO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ IDNO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ IDNO:51, SEQ ID NO:53, SEQ ID NO:55, SEQ ID NO:57, SEQ ID NO:59, SEQ IDNO:61, SEQ ID NO:63, SEQ ID NO:65, SEQ ID NO:67, SEQ ID NO:69, SEQ IDNO:71, SEQ ID NO:73, SEQ ID NO:75, SEQ ID NO:77, SEQ ID NO:79, SEQ IDNO:81, SEQ ID NO:83, SEQ ID NO:85, SEQ ID NO:87, SEQ ID NO:89, SEQ IDNO:91, SEQ ID NO:93, SEQ ID NO:95, SEQ ID NO:97, SEQ ID NO:99, SEQ IDNO:101, SEQ ID NO:103, SEQ ID NO:105, SEQ ID NO:107, SEQ ID NO:109, SEQID NO:111, SEQ ID NO:113, SEQ ID NO:115, SEQ ID NO:117, SEQ ID NO:119,SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ IDNO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147,SEQ ID NO:149, SEQ ID NO:151, SEQ ID NO:153, SEQ ID NO:155, SEQ IDNO:157, SEQ ID NO:159, SEQ ID NO:161, SEQ ID NO:163, SEQ ID NO:165, SEQID NO:167, SEQ ID NO:169, SEQ ID NO:171, SEQ ID NO:173, SEQ ID NO:175,SEQ ID NO:177, SEQ ID NO:179, SEQ ID NO:181, SEQ ID NO:183, SEQ IDNO:185, SEQ ID NO:187, SEQ ID NO:189, SEQ ID NO:191, SEQ ID NO:193, SEQID NO:195, SEQ ID NO:197, SEQ ID NO:199, SEQ ID NO:201, SEQ ID NO:203,SEQ ID NO:205, SEQ ID NO:207, SEQ ID NO:209, SEQ ID NO:211, SEQ IDNO:213, SEQ ID NO:215, SEQ ID NO:217, SEQ ID NO:219, SEQ ID NO:221, SEQID NO:223, SEQ ID NO:225, SEQ ID NO:227, SEQ ID NO:229, SEQ ID NO:231,SEQ ID NO:233, SEQ ID NO:235, SEQ ID NO:237, SEQ ID NO:239, SEQ IDNO:241, SEQ ID NO:243, SEQ ID NO:245, SEQ ID NO:247, SEQ ID NO:249, SEQID NO:251, SEQ ID NO:253, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259,SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ IDNO:269, SEQ ID NO:271, SEQ ID NO:273, SEQ ID NO:275, SEQ ID NO:277, SEQID NO:279, SEQ ID NO:281, SEQ ID NO:283, SEQ ID NO:285, SEQ ID NO:287,SEQ ID NO:289, SEQ ID NO:291, SEQ ID NO:293, SEQ ID NO:295, SEQ IDNO:297, SEQ ID NO:299, SEQ ID NO:301, SEQ ID NO:303, SEQ ID NO:305, SEQID NO:307, SEQ ID NO:309, SEQ ID NO:311, SEQ ID NO:313, SEQ ID NO:315,SEQ ID NO:317, SEQ ID NO:319, SEQ ID NO:321, SEQ ID NO:323, SEQ IDNO:325, SEQ ID NO:327, SEQ ID NO:329, SEQ ID NO:331, SEQ ID NO:333, SEQID NO:335, SEQ ID NO:337, SEQ ID NO:339, SEQ ID NO:341, SEQ ID NO:343,SEQ ID NO:345, SEQ ID NO:347, SEQ ID NO:349, SEQ ID NO:351, SEQ IDNO:353, SEQ ID NO:355, SEQ ID NO:357, SEQ ID NO:359, SEQ ID NO:361, SEQID NO:363, SEQ ID NO:365, SEQ ID NO:367, SEQ ID NO:369, SEQ ID NO:371,SEQ ID NO:373, SEQ ID NO:375, SEQ ID NO:377, SEQ ID NO:379, SEQ IDNO:381, SEQ ID NO:383, SEQ ID NO:385, SEQ ID NO:387, SEQ ID NO:389, SEQID NO:391, SEQ ID NO:393, SEQ ID NO:395, SEQ ID NO:397, SEQ ID NO:399,SEQ ID NO:401, SEQ ID NO:403, SEQ ID NO:405, SEQ ID NO:407, SEQ IDNO:409, SEQ ID NO:411, SEQ ID NO:413, SEQ ID NO:415, SEQ ID NO:417, SEQID NO:419, SEQ ID NO:421, SEQ ID NO:423, SEQ ID NO:425, SEQ ID NO:427,SEQ ID NO:429, SEQ ID NO:431, SEQ ID NO:433, SEQ ID NO:435, SEQ IDNO:437, SEQ ID NO:439, SEQ ID NO:441, SEQ ID NO:443, SEQ ID NO:445, SEQID NO:447, SEQ ID NO:449, SEQ ID NO:451, SEQ ID NO:453, SEQ ID NO:455,SEQ ID NO:457, SEQ ID NO:459, SEQ ID NO:461, SEQ ID NO:463, SEQ IDNO:465, SEQ ID NO:467, SEQ ID NO:469, SEQ ID NO:471, SEQ ID NO:473, SEQID NO:475, SEQ ID NO:477, SEQ ID NO:479, SEQ ID NO:481, SEQ ID NO:483,SEQ ID NO:485, SEQ ID NO:487, SEQ ID NO:489, SEQ ID NO:491, SEQ IDNO:493, SEQ ID NO:495 or SEQ ID NO:497, and as described herein and inTables 1, 2 and 3, and the Sequence Listing (all of these sequences are“exemplary polynucleotides of the invention”), and enzymatically activesubsequences (fragments) thereof, over a region of between about 10 to2500, or more residues, or the full length of a cDNA, transcript (mRNA)or gene. Nucleic acids of the invention includes those encoding apolypeptide of this invention, having at least 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% or more, or 100% (complete) sequence identity to anexemplary polypeptide of the invention, which includes, e.g., SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12,SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32,SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42,SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52,SEQ ID NO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62,SEQ ID NO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72,SEQ ID NO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82,SEQ ID NO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92,SEQ ID NO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102,SEQ ID NO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ IDNO:112, SEQ ID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130,SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ IDNO:140, SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQID NO:150, SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158,SEQ ID NO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ IDNO:168, SEQ ID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQID NO:178, SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186,SEQ ID NO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ IDNO:196, SEQ ID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQID NO:206, SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214,SEQ ID NO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ IDNO:224, SEQ ID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQID NO:234, SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242,SEQ ID NO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ IDNO:252, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270,SEQ ID NO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ IDNO:280, SEQ ID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQID NO:290, SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298,SEQ ID NO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ IDNO:308, SEQ ID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQID NO:318, SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326,SEQ ID NO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ IDNO:336, SEQ ID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQID NO:346, SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354,SEQ ID NO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ IDNO:364, SEQ ID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQID NO:374, SEQ ID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382,SEQ ID NO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ IDNO:392, SEQ ID NO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQID NO:402, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410,SEQ ID NO:412, SEQ ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ IDNO:420, SEQ ID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438,SEQ ID NO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ IDNO:448, SEQ ID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466,SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ IDNO:476, SEQ ID NO:478, SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQID NO:486, SEQ ID NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494,SEQ ID NO:496 or SEQ ID NO:498, including the sequences described hereinand in Tables 1, 2 and 3, below, and in the Sequence Listing (all ofthese sequences are “exemplary enzymes/polypeptides of the invention”),and enzymatically active subsequences (fragments) thereof.

Tables 1, 2 and 3, below, are charts describing selected characteristicsof exemplary nucleic acids and polypeptides of the invention, includingsequence identity comparison of the exemplary sequences to publicdatabases.

Table 1, below, describes the assigned activity (as determined byexperimental data, see Examples 1-19, below) of the exemplarypolypeptides (encoded by the exemplary polynucleotides) of theinvention. Table 1 further indicates whether the polynucleotide(encoding a polypeptide) of the invention is a clone (a genomic sequenceisolated from the original source, as described in Table 2) or is asubclone (where the clone is manipulated by, e.g. removal of a nativesignal sequence, addition of a start Methionine, addition of a tag,etc). Table 1 also indicates the clone and subclone relationship, e.g.which subclone was derived from which clone. For aid in reading Table 1,for example, Columns 1 and 4, rows 1 and 2, indicate that SEQ ID NO:34(encoded by SEQ ID NO:33) is a clone with the corresponding subclonebeing SEQ ID NO:464 (encoded by SEQ ID NO:463), denoted as“Clone/subclone pair 1”.

Table 2, below, indicates the source from which the exemplary nucleicacids and polypeptides of the invention were first derived. Table 2,below, also indicates the “Signalp Cleavage Site” for the exemplaryenzyme's signal sequence (or “signal peptide”, or SP), as determined bythe paradigm Signalp, as discussed below (see Nielsen (1997), infra);the “Predicted Signal Sequence” is listed from the amino terminal to thecarboxy terminal, for example, for the polypeptide SEQ ID NO:42, thesignal peptide is “MPFCRTLLAVSLGLLITGQAPLYA” (amino acids 1-24 of SEQ IDNO:42).

Table 3, below describes selected characteristics of exemplary nucleicacids and polypeptides of the invention, including sequence identitycomparison of the exemplary sequences to public databases. To furtheraid in reading Table 3, for example, the first row, labeled “SEQ IDNO:”, the numbers “1, 2” represent the exemplary polypeptide of theinvention having a sequence as set forth in SEQ ID NO:2, encoded by,e.g., SEQ ID NO:1. All sequences described in Table 2 (all the exemplarysequences of the invention) have been subject to a BLAST search (asdescribed in detail, below) against two sets of databases. The firstdatabase set is available through NCBI (National Center forBiotechnology Information). All results from searches against thesedatabases are found in the columns entitled “NR Description”, “NRAccession Code”, “NR Evalue” or “NR Organism”. “NR” refers to theNon-Redundant nucleotide database maintained by NCBI. This database is acomposite of GenBank, GenBank updates, and EMBL updates. The entries inthe column “NR Description” refer to the definition line in any givenNCBI record, which includes a description of the sequence, such as thesource organism, gene name/protein name, or some description of thefunction of the sequence. The entries in the column “NR Accession Code”refer to the unique identifier given to a sequence record. The entriesin the column “NR Evalue” refer to the Expect value (Evalue), whichrepresents the probability that an alignment score as good as the onefound between the query sequence (the sequences of the invention) and adatabase sequence would be found in the same number of comparisonsbetween random sequences as was done in the present BLAST search. Theentries in the column “NR Organism” refer to the source organism of thesequence identified as the closest BLAST hit. The second set ofdatabases is collectively known as the GENESEQ™ database, which isavailable through Thomson Derwent (Philadelphia, Pa.). All results fromsearches against this database are found in the columns entitled“GENESEQ™ Protein Description”, “GENESEQ™ Protein Accession Code”,“Evalue”, “GENESEQ™ DNA Description”, “GENESEQ™ DNA Accession Code” or“Evalue”. The information found in these columns is comparable to theinformation found in the NR columns described above, except that it wasderived from BLAST searches against the GENESEQ™ database instead of theNCBI databases. In addition, this table includes the column “PredictedEC No.”. An EC number is the number assigned to a type of enzymeaccording to a scheme of standardized enzyme nomenclature developed bythe Enzyme Commission of the Nomenclature Committee of the InternationalUnion of Biochemistry and Molecular Biology (IUBMB). The results in the“Predicted EC No.” column are determined by a BLAST search against theKegg (Kyoto Encyclopedia of Genes and Genomes) database. If the topBLAST match has an Evalue equal to or less than e-6, the EC numberassigned to the top match is entered into the table. The EC number ofthe top hit is used as a guide to what the EC number of the sequence ofthe invention might be. The columns “Query DNA Length” and “QueryProtein Length” refer to the number of nucleotides or the number aminoacids, respectively, in the sequence of the invention that was searchedor queried against either the NCBI or GENESEQ™ databases. The columns“Subject DNA Length” and “Subject Protein Length” refer to the number ofnucleotides or the number amino acids, respectively, in the sequence ofthe top match from the BLAST searches. The results provided in thesecolumns are from the search that returned the lower Evalue, either fromthe NCBI databases or the Geneseq database. The columns “% ID Protein”and “% ID DNA” refer to the percent sequence identity between thesequence of the invention and the sequence of the top BLAST match. Theresults provided in these columns are from the search that returned thelower Evalue, either from the NCBI databases or the GENESEQ™ database.

TABLE 1 Sequence type Clone/subclone pair SEQ ID NO: Activity (Clone orsubclone) 1 33, 34 Racemase Clone 1 463, 464 Racemase Subclone 2 131,132 Racemase Clone 2 457, 458 Racemase Subclone 3 13, 14 Racemase Clone3 387, 388 Racemase Subclone 4 25, 26 Racemase Clone 4 389, 390 RacemaseSubclone 5 23, 24 Racemase Clone 5 391, 392 Racemase Subclone 6 61, 62Racemase Clone 6 411, 412 Racemase Subclone (leaderless) 6 489, 490Racemase Subclone 7 297, 298 Racemase Clone 7 467, 468 Racemase Subclone8 11, 12 Racemase Clone 8 397, 398 Racemase Subclone 9 311, 312 RacemaseClone 9 469, 470 Racemase Subclone 10 3, 4 Racemase Clone 10 393, 394Racemase Subclone 11 49, 50 Racemase Clone 11 423, 424 Racemase Subclone12 367, 368 Racemase Clone 12 471, 472 Racemase Subclone 13 41, 42Racemase Clone 13 399, 400 Racemase Subclone (leaderless) 13 491, 492Racemase Subclone 14 43, 44 Racemase Clone 14 431, 432 Racemase Subclone15 45, 46 Racemase Clone 15 409, 410 Racemase Subclone (leaderless) 15495, 496 Racemase Subclone 16 47, 48 Racemase Clone 16 407, 408 RacemaseSubclone (leaderless) 16 493, 494 Racemase Subclone 17 287, 288 RacemaseClone 17 453, 454 Racemase Subclone 18 35, 36 Racemase Clone 18 385, 386Racemase Subclone 19 51, 52 Racemase Clone 19 401, 402 Racemase Subclone(leaderless) 19 497, 498 Racemase Subclone 20 53, 54 Racemase Clone 20403, 404 Racemase Subclone (leaderless) 20 427, 428 Racemase Subclone 2155, 56 Racemase Clone 21 433, 434 Racemase Subclone 22 107, 108 RacemaseClone 22 473, 474 Racemase Subclone 23 57, 58 Racemase Clone 23 405, 406Racemase Subclone (leaderless) 23 429, 430 Racemase Subclone 24 109, 110Racemase Clone 24 415, 416 Racemase Subclone 25 121, 122 Racemase Clone25 425, 426 Racemase Subclone 26 301, 302 Racemase Clone 26 477, 478Racemase Subclone 27 123, 124 Racemase Clone 27 439, 440 RacemaseSubclone 28 125, 126 Racemase Clone 28 461, 462 Racemase Subclone 29299, 300 Racemase Clone 29 475, 476 Racemase Subclone 30 111, 112Racemase Clone 30 417, 418 Racemase Subclone 31 119, 120 Racemase Clone31 459, 460 Racemase Subclone 32 113, 114 Racemase Clone 32 435, 436Racemase Subclone 33 115, 116 Racemase Clone 33 419, 420 RacemaseSubclone 34 117, 118 Racemase Clone 34 421, 422 Racemase Subclone 35223, 224 Racemase Clone 35 441, 442 Racemase Subclone 36 217, 218Racemase Clone 36 443, 444 Racemase Subclone 37 233, 234 Racemase Clone37 445, 446 Racemase Subclone 38 243, 244 Racemase Clone 38 447, 448Racemase Subclone 39 247, 248 Racemase Clone 39 449, 450 RacemaseSubclone 40 273, 274 Racemase Clone 40 451, 452 Racemase Subclone 41105, 106 Racemase Clone 41 465, 466 Racemase Subclone 42 103, 104Racemase Clone 42 437, 438 Racemase Subclone 43 7, 8 Racemase Clone 43413, 414 Racemase Subclone 44  9, 10 Racemase Clone 44 395, 396 RacemaseSubclone 45 129, 130 Racemase Clone 45 455, 456 Racemase Subclone 379,380 Epimerase Clone 381, 382 Epimerase Clone 369, 370 Epimerase Clone375, 376 Epimerase Clone 383, 384 Isomerase Clone 373, 374 EpimeraseClone 371, 372 Epimerase Clone 377, 378 Epimerase Clone 17, 18 RacemaseClone 19, 20 Racemase Clone 15, 16 Racemase Clone 29, 30 Racemase Clone27, 28 Racemase Clone 255, 256 Racemase Clone 321, 322 Racemase Clone323, 324 Racemase Clone 327, 328 Racemase Clone 307, 308 Racemase Clone303, 304 Racemase Clone 309, 310 Racemase Clone 305, 306 Racemase Clone21, 22 Racemase Clone 479, 480 Racemase Clone 313, 314 Racemase Clone315, 316 Racemase Clone 1, 2 Racemase Clone 85, 86 Racemase Clone 87, 88Racemase Clone 89, 90 Racemase Clone 91, 92 Racemase Clone 93, 94Racemase Clone 99, 100 Racemase Clone 77, 78 Racemase Clone 331, 332Racemase Clone 345, 346 Racemase Clone 347, 348 Racemase Clone 333, 334Racemase Clone 325, 326 Racemase Clone 319, 320 Racemase Clone 335, 336Racemase Clone 349, 350 Racemase Clone 339, 340 Racemase Clone 341, 342Racemase Clone 343, 344 Racemase Clone 355, 356 Racemase Clone 353, 354Racemase Clone 351, 352 Racemase Clone 317, 318 Racemase Clone 329, 330Racemase Clone 167, 168 Racemase Clone 213, 214 Racemase Clone 285, 286Racemase Clone 289, 290 Racemase Clone 37, 38 Racemase Clone 39, 40Racemase Clone 483, 484 Racemase Clone 485, 486 Racemase Clone 487, 488Racemase Clone 31, 32 Racemase Clone 101, 102 Racemase Clone 169, 170Racemase Clone 171, 172 Racemase Clone 59, 60 Racemase Clone 135, 136Racemase Clone 173, 174 Racemase Clone 137, 138 Racemase Clone 337, 338Racemase Clone 357, 358 Racemase Clone 359, 360 Racemase Clone 361, 362Racemase Clone 363, 364 Racemase Clone 365, 366 Racemase Clone 175, 176Racemase Clone 177, 178 Racemase Clone 179, 180 Racemase Clone 181, 182Racemase Clone 143, 144 Racemase Clone 187, 188 Racemase Clone 189, 190Racemase Clone 133, 134 Racemase Clone 145, 146 Racemase Clone 481, 482Racemase Clone 63, 64 Racemase Clone 193, 194 Racemase Clone 153, 154Racemase Clone 155, 156 Racemase Clone 195, 196 Racemase Clone 157, 158Racemase Clone 159, 160 Racemase Clone 161, 162 Racemase Clone 163, 164Racemase Clone 165, 166 Racemase Clone 65, 66 Racemase Clone 147, 148Racemase Clone 149, 150 Racemase Clone 191, 192 Racemase Clone 151, 152Racemase Clone 71, 72 Racemase Clone 69, 70 Racemase Clone 73, 74Racemase Clone 75, 76 Racemase Clone 95, 96 Racemase Clone 97, 98Racemase Clone 79, 80 Racemase Clone 81, 82 Racemase Clone 83, 84Racemase Clone 67, 68 Racemase Clone 183, 184 Racemase Clone 139, 140Racemase Clone 141, 142 Racemase Clone 185, 186 Racemase Clone 197, 198Racemase Clone 199, 200 Racemase Clone 201, 202 Racemase Clone 203, 204Racemase Clone 205, 206 Racemase Clone 207, 208 Racemase Clone 209, 210Racemase Clone 211, 212 Racemase Clone 219, 220 Racemase Clone 221, 222Racemase Clone 225, 226 Racemase Clone 227, 228 Racemase Clone 229, 230Racemase Clone 231, 232 Racemase Clone 235, 236 Racemase Clone 237, 238Racemase Clone 239, 240 Racemase Clone 241, 242 Racemase Clone 293, 294Racemase Clone 245, 246 Racemase Clone 295, 296 Racemase Clone 249, 250Racemase Clone 251, 252 Racemase Clone 253, 254 Racemase Clone 257, 258Racemase Clone 259, 260 Racemase Clone 261, 262 Racemase Clone 263, 264Racemase Clone 265, 266 Racemase Clone 267, 268 Racemase Clone 269, 270Racemase Clone 271, 272 Racemase Clone 275, 276 Racemase Clone 277, 278Racemase Clone 279, 280 Racemase Clone 281, 282 Racemase Clone 283, 284Racemase Clone 291, 292 Racemase Clone 215, 216 Racemase Clone 5, 6Racemase Clone 127, 128 Racemase Clone

TABLE 2  SEQ ID NO: Source Signalp Cleavage SitePredicted Signal Sequence 1, 2 Aquifex aeolicus 3, 4 Aquifex aeolicus5, 6 Bacteria 7, 8 Bacteria 9, 10 Bacteria 11, 12 Unknown 13, 14 Unknown15, 16 Unknown 17, 18 Unknown 19, 20 Unknown 21, 22 Unknown 23, 24Unknown 25, 26 Unknown 27, 28 Pelagibacter ubique 29, 30 Unknown 31, 32Unknown 33, 34 Unknown 35, 36 Unknown 37, 38 Unknown 39, 40 Unknown41, 42 Unknown Probability: 0.999 AA1: MPFCRTLLAVSLGLLITGQAPLYA24 AA2: 25 43, 44 Unknown Probability: 0.999 AA1:MPFCRTLLAVSLGLLITGQAPLYA 24 AA2: 25 45, 46 UnknownProbability: 1.000 AA1: MPFSRTLLAVSLGLLITGQAPLYA 24 AA2: 25 47, 48Unknown Probability: 1.000 AA1: MPFSRTLLAVSLGLLITGQAPLYA 24 AA2: 2549, 50 Unknown Probability: 1.000 AA1: MPFCRTLLAASLALLITGQAPLYA24 AA2: 25 51, 52 Unknown Probability: 1.000 AA1:MPFRRTLLALSLGLVLWQGQVHA 23 AA2: 24 53, 54 UnknownProbability: 1.000 AA1: MPFCRTLLALSLGLVLWQGQAHA 23 AA2: 24 55, 56Unknown Probability: 1.000 AA1: MPFCRTLLALSLGLVLWQGQVHA 23 AA2: 2457, 58 Unknown Probability: 1.000 AA1: MPFSRTLLAASLALLITGQAPLYA24 AA2: 25 59, 60 Unknown Probability: 1.000 AA1:MPFCRTLLAASLALLITGQAPLYA 24 AA2: 25 61, 62 UnknownProbability: 0.999 AA1: MFTMIFMKKKFCLLFATIILFITCLC 33 AA2: 34 FLLKSVS63, 64 Unknown Probability: 1.000 AA1: MPFRRTLLAASLALLITGLAPLYA24 AA2: 25 65, 66 Unknown Probability: 1.000 AA1:MPFPRTLLAASLALLITGQAPLYA 24 AA2: 25 67, 68 UnknownProbability: 1.000 AA1: MPFRRTLLAASLALLVTAQAPLYA 24 AA2: 25 69, 70Unknown Probability: 1.000 AA1: MPFCRTLLAASLALLITGQAPLYA 24 AA2: 2571, 72 Unknown Probability: 1.000 AA1: MPFRRTLLAASLALLITGQAPLYA24 AA2: 25 73, 74 Unknown Probability: 1.000 AA1:MPFCRTLLAASLALLITGQAPLYA 24 AA2: 25 75, 76 UnknownProbability: 1.000 AA1: MPFPRTLLAASLALLITGQAPLYA 24 AA2: 25 77, 78Unknown Probability: 1.000 AA1: MPFRRTLLAASLALLITGQAPLYA 24 AA2: 2579, 80 Unknown Probability: 1.000 AA1: MPFCRTLLAASLALLITGQAPLYA24 AA2: 25 81, 82 Unknown Probability: 1.000 AA1:MPFCRTLLAASLALLITGQAPLYA 24 AA2: 25 83, 84 UnknownProbability: 1.000 AA1: MPFCRTLLAASLALLITGQAPLYA 24 AA2: 25 85, 86Unknown Probability: 1.000 AA1: MPFSRTLLAASLALLITGQAPLFA 24 AA2: 2587, 88 Unknown Probability: 1.000 AA1: MPFCRTLLAASLALLITGQAPLYA24 AA2: 25 89, 90 Unknown Probability: 1.000 AA1:MPFRRTLLAASLALLITGQAPLFA 24 AA2: 25 91, 92 UnknownProbability: 1.000 AA1: MPFCRTLLAASLALLITGQAPLYA 24 AA2: 25 93, 94Unknown Probability: 1.000 AA1: MPFRRTLLAASLALLITGQAPLYA 24 AA2: 2595, 96 Unknown Probability: 1.000 AA1: MPFPRTLLAASLALLITGQAPLYA24 AA2: 25 97, 98 Unknown Probability: 1.000 AA1:MPFRRTLLAASLALLITGQAPLYA 24 AA2: 25 99, 100 UnknownProbability: 1.000 AA1: MPFCRTLLAASLALLITGQAPLYA 24 AA2: 25 101, 102Unknown 103, 104 Unknown Probability: 0.826 AA1: MKNNKCIAILGGMGPQASS19 AA2: 20 105, 106 Unknown 107, 108 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 109, 110 UnknownProbability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2:22 111, 112 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2:22 113, 114 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2:22 115, 116 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2:22 117, 118 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2:22 119, 120 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2:22 121, 122 Unknown123, 124 Unknown 125, 126 Unknown 127, 128 Unknown 129, 130 Unknown131, 132 Unknown 133, 134 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 135, 136 UnknownProbability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 137, 138Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22139, 140 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 141, 142 Unknown Probability: 1.000 AA1:MHKKTLLATLIFGLLAGQAVA 21 AA2:22 143, 144 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 145, 146 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22 147, 148Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22149, 150 Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA21 AA2: 22 151, 152 Unknown Probability: 1.000 AA1:MHKKTLLATLVFGLLAGQAVA 21 AA2:22 153, 154 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 155, 156 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22 157, 158Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22159, 160 Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA21 AA2: 22 161, 162 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2:22 163, 164 Unknown 165, 166 Unknown167, 168 Unknown 169, 170 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 171, 172 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2:22 173, 174 UnknownProbability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 175, 176Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22177, 178 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 179, 180 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 181, 182 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2:22 183, 184 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22 185, 186Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22187, 188 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 189, 190 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 191, 192 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2:22 193, 194 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 195, 196Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22197, 198 Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA21 AA2: 22 199, 200 Unknown Probability: 1.000 AA1:MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 201, 202 UnknownProbability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 203, 204Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22205, 206 Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA21 AA2: 22 207, 208 Unknown Probability: 1.000 AA1:MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 209, 210 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 211, 212Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2:22 213, 214Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22215, 216 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 217, 218 Unknown Probability: 1.000 AA1:MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 219, 220 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22 221, 222Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22223, 224 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 225, 226 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 227, 228 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 229, 230Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22231, 232 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 233, 234 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 235, 236 UnknownProbability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 237, 238Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22239, 240 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 241, 242 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 243, 244 UnknownProbability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 245, 246Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22247, 248 Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA21 AA2: 22 249, 250 Unknown Probability: 1.000 AA1:MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 251, 252 UnknownProbability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22 253, 254Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA 21 AA2: 22255, 256 Unknown 257, 258 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 259, 260 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22 261, 262Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22263, 264 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 265, 266 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 267, 268 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22 269, 270Unknown Probability: 1.000 AA1: MHKKTLLATLVLGLLAGQAVA 21 AA2: 22271, 272 Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA21 AA2: 22 273, 274 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 275, 276 UnknownProbability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22 277, 278Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22279, 280 Unknown Probability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA21 AA2: 22 281, 282 Unknown Probability: 1.000 AA1:MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 283, 284 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 285, 286Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22287, 288 Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA21 AA2: 22 289, 290 Unknown Probability: 1.000 AA1:MHKKTLLATLILGLLAGQAVA 21 AA2: 22 291, 292 UnknownProbability: 1.000 AA1: MHKKTLLATLVFGLLAGQAVA 21 AA2: 22 293, 294Unknown Probability: 1.000 AA1: MHKKTLLATLILGLLAGQAVA 21 AA2: 22295, 296 Unknown Probability: 1.000 AA1: MHKKTLLATLIFGLLAGQAVA21 AA2: 22 297, 298 Unknown 299, 300 Unknown Probability: 1.000 AA1:MPFTRTVLALSLGLVLLQSQVHA 23 AA2: 24 301, 302 UnknownProbability: 1.000 AA1: MKFTPTLLAVALAGCLSTQVQA 22 AA2: 23 303, 304Unknown 305, 306 Unknown 307, 308 Unknown 309, 310 Unknown 311, 312Unknown 313, 314 Unknown 315, 316 Unknown 317, 318 Unknown 319, 320Unknown 321, 322 Unknown 323, 324 Unknown 325, 326 Unknown 327, 328Unknown 329, 330 Unknown 331, 332 Unknown 333, 334 Unknown 335, 336Unknown 337, 338 Unknown 339, 340 Unknown 341, 342 Unknown 343, 344Unknown 345, 346 Unknown 347, 348 Unknown 349, 350 Unknown 351, 352Unknown 353, 354 Unknown 355, 356 Unknown 357, 358 Unknown 359, 360Unknown 361, 362 Unknown 363, 364 Unknown 365, 366 Unknown 367, 368Pseudomonas straminea ATCC 33636 369, 370 Unknown 371, 372 Unknown373, 374 Unknown 375, 376 Unknown 377, 378 Unknown 379, 380 Unknown381, 382 Unknown 383, 384 Unknown 385, 386 Unknown 387, 388 Unknown389, 390 Unknown 391, 392 Unknown 393, 394 Unknown 395, 396 Unknown397, 398 Unknown 399, 400 Unknown 401, 402 Unknown 403, 404 Unknown405, 406 Unknown 407, 408 Unknown 409, 410 Unknown 411, 412 Unknown413, 414 Unknown 415, 416 Unknown 417, 418 Unknown 419, 420 Unknown421, 422 Unknown 423, 424 Unknown 425, 426 Unknown 427, 428 UnknownProbability: 1.000 AA1: MPFCRTLLALSLGLVLWQGQAHA 23 AA2: 24 429, 430Unknown Probability: 1.000 AA1: MPFSRTLLAASLALLITGQAPLYA 24 AA2: 25431, 432 Unknown 433, 434 Unknown 435, 436 Unknown 437, 438 UnknownProbability: 0.826 AA1: MKNNKCIAILGGMGPQASS 19 AA2: 20 439, 440 Unknown441, 442 Unknown 443, 444 Unknown 445, 446 Unknown 447, 448 Unknown449, 450 Unknown 451, 452 Unknown 453, 454 Unknown 455, 456 Unknown457, 458 Unknown Probability: 0.742 AA1: MARVVLRWARSAYIRITTGSHALF30 AA2: 31 ADATLA 459, 460 Unknown 461, 462 Unknown 463, 464 Unknown465, 466 Unknown 467, 468 Unknown 469, 470 Unknown 471, 472 Unknown473, 474 Unknown 475, 476 Unknown 477, 478 Unknown 479, 480 Unknown481, 482 Unknown 483, 484 Unknown 485, 486 Unknown 487, 488 Unknown

TABLE 3 Geneseq NR Geneseq Protein Accession NR Protein Accession SEQ IDNO: NR Description Code Evalue Organism Description Code Evalue 1, 2hypothetical 15605814 1.00E−128 Aquifex Prokaryotic ABU25646 3.00E−47protein [Aquifex aeolicus essential aeolicus]. gene #34740. 3, 4 alanineracemase 15606873 0 Aquifex Aquifex ABB06296 1.00E−147 [Aquifexaeolicus]. aeolicus pyrophilus heat resistant alanine racemase encodingDNA. 5, 6 hypothetical 29832668 1.00E−101 Streptomyces ProkaryoticABU19499 5.00E−37 protein SAV6126 avermitilis essential [StreptomycesMA- gene avermitilis MA- 4680 #34740. 4680] 7, 8 hypothetical 29830835 0Streptomyces Propionibacterium ABM54358 2.00E−53 protein SAV4292avermitilis acnes [Streptomyces MA- predicted avermitilis MA- 4680 ORF-4680] encoded polypeptide #300. 9, alanine racemase 21223124 1.00E−166Streptomyces Prokaryotic ABU34223 6.00E−90 10 [Streptomyces coelicoloressential coelicolor A3(2)] A3(2) gene #34740. 11, alanine racemase86748627 1.00E−106 Rhodopseudomonas Pseudomonas ABO84274 4.00E−50 12[Rhodopseudomonas palustris aeruginosa palustris HaA2 polypeptide HaA2]#3. 13, alanine racemase 56477426 1.00E−128 Azoarcus ProkaryoticABU41398 1.00E−110 14 [Azoarcus sp. sp. EbN1 essential EbN1] gene#34740. 15, Aspartate 1.49E+08 4.00E−93 Marinobacter Klebsiella ABO655031.00E−71 16 racemase algicola pneumoniae [Marinobacter DG893 polypeptidealgicola DG893] seqid 7178. 17, glutamate 1.11E+08 1.00E−106 CytophagaProkaryotic ABU25174 2.00E−44 18 racemase hutchinsonii essential[Cytophaga ATCC gene hutchinsonii ATCC 33406 #34740. 33406] 19,glutamate 39998014 1.00E−74 Geobacter M. xanthus ABM90755 2.00E−67 20racemase sulfurreducens protein [Geobacter PCA sequence, sulfurreducensseq id PCA] 9726. 21, Putative 33596748 5.00E−40 Bordetella ThermococcusADN46910 5.00E−27 22 decarboxylase parapertussis kodakaraensis[Bordetella 12822 KOD1 parapertussis protein 12822] sequence SeqID4. 23,alanine racemase 1.46E+08 0 Enterobacter Enterobacter AEH63094 0 24[Enterobacter sp. sp. cloacae 638] 638 protein amino acid sequence - SEQID 5666. 25, alanine racemase 1.46E+08 0 Enterobacter EnterobacterAEH63094 0 26 [Enterobacter sp. sp. cloacae 638] 638 protein amino acidsequence - SEQ ID 5666. 27, hypothetical 71083067 1.00E−121 CandidatusProkaryotic ABU24290 2.00E−31 28 protein Pelagibacter essentialSAR11_0361 ubique gene [Candidatus HTCC1062 #34740. Pelagibacter ubiqueHTCC1062] 29, putative proline 1.49E+08 1.00E−130 Brucella PseudomonasABO82134 1.00E−127 30 racemase ovis aeruginosa [Brucella ovis ATCCpolypeptide ATCC 25840] 25840 #3. 31, proline racemase, 1.19E+081.00E−109 Stappia Acinetobacter ADA35228 1.00E−105 32 putative [Stappiaaggregata baumannii aggregata IAM IAM protein 12614] 12614 #19.gi|118435940|gb|EAV42584.1| proline racemase, putative [Stappiaaggregata IAM 12614] 33, alanine racemase 1.11E+08 1.00E−124Mesorhizobium Prokaryotic ABU38829 3.00E−46 34 [Mesorhizobium sp. BNC1essential sp. BNC1] gene #34740. 35, putative alanine 90418439 6.00E−49Aurantimonas Achromobacter AEH19277 5.00E−47 36 racemase sp.xylosoxidans [Aurantimonas sp. SI85-9A1 DTA SI85-9A1] SEQ ID NOgi|90338111|gb|EAS51762.1| 6. putative alanine racemase [Aurantimonassp. SI85-9A1] 37, Alanine racemase 1.45E+08 1.00E−38 MagnetospirillumAquifex ABB06296 2.00E−20 38 [Magnetospirillum gryphiswaldensepyrophilus gryphiswaldense MSR-1 heat MSR-1] resistant alanine racemaseencoding DNA. 39, putative proline 91779222 2.00E−36 BurkholderiaProkaryotic ABU21813 5.00E−37 40 racemase xenovorans essential[Burkholderia LB400 gene xenovorans #34740. LB400] 41, alanine racemase1.49E+08 0 Pseudomonas T. maritima AED11803 0 42 [Pseudomonas putida F1D-alanine- putida F1] D-alanine ligase. 43, alanine racemase 1.49E+08 0Pseudomonas T. maritima AED11803 0 44 [Pseudomonas putida F1 D-alanine-putida F1] D-alanine ligase. 45, alanine racemase 1.49E+08 0 PseudomonasT. maritima AED11803 0 46 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 47, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 48 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 49, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 50 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 51, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 1.00E−180 52 [Pseudomonas putida D-alanine- putidaGB-1] GB-1 D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanineracemase [Pseudomonas putida GB-1] 53, alanine racemase 1.26E+081.00E−180 Pseudomonas T. maritima AED11803 1.00E−180 54 [Pseudomonasputida D-alanine- putida GB-1] GB-1 D-alaninegi|126314851|gb|EAZ66019.1| ligase. alanine racemase [Pseudomonas putidaGB-1] 55, alanine racemase 1.26E+08 1.00E−180 Pseudomonas T. maritimaAED11803 1.00E−179 56 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 57, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 58 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 59, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 60 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 61, alanine racemase 1.48E+08 7.00E−19 FusobacteriumAquifex ABB06296 1.00E−12 62 [Fusobacterium nucleatum pyrophilusnulceatum subsp. subsp. heat polymorphum polymorphum resistant ATCC10953] ATCC alanine 10953 racemase encoding DNA. 63, alanine racemase1.49E+08 0 Pseudomonas T. maritima AED11803 0 64 [Pseudomonas putida F1D-alanine- putida F1] D-alanine ligase. 65, alanine racemase 1.49E+08 0Pseudomonas T. maritima AED11803 0 66 [Pseudomonas putida F1 D-alanine-putida F1] D-alanine ligase. 67, alanine racemase 1.26E+08 0 PseudomonasT. maritima AED11803 0 68 [Pseudomonas putida D-alanine- putida GB-1]GB-1 D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 69, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 70 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 71, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 72 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 73, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 74 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 75, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 76 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 77, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 78 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 79, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 80 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 81, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 82 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 83, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 84 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 85, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 86 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 87, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 88 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 89, alanine racemase 26990430 0 Pseudomonas T.maritima AED11803 0 90 [Pseudomonas putida D-alanine- putida KT2440]KT2440 D-alanine ligase. 91, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 92 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 93, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 94 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 95, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 96 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 97, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 98 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 99, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 100 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 101, alanine racemase 1580 2.00E−58 EscherichiaProkaryotic ABU45089 2.00E−58 102 2, catabolic 1412 coli essential[Escherichia coli O157:H7 gene O157:H7 EDL933 #34740. EDL933]. 103,aspartate 1452 8.00E−29 Pyrococcus Thermococcus ADN46207 2.00E−25 104racemase 1575 abyssi kodakaraensis [Pyrococcus KOD1 abyssi]. proteinsequence SeqID4. 105, proline racemase 1.24E+08 1.00E−150 MicroscillaBacterial ADS22995 1.00E−128 106 [Microscilla marina marina polypeptideATCC 23134] ATCC #10001. gi|123984081|gb|EAY24454.1| 23134 prolineracemase [Microscilla marina ATCC 23134] 107, alanine racemase 1.18E+080 Aeromonas Empedobacter AED10581 1.00E−131 108 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 109, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 110[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO:3. 111, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 112 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 113, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 114 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 115, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 116 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.117, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 118 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 119, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 120 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 121, alanine racemase1.49E+08 1.00E−136 Pseudomonas T. maritima AED11803 1.00E−137 122[Pseudomonas putida F1 D-alanine- putida F1] D-alanine ligase. 123,alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−127124 [Aeromonas hydrophila brevis hydrophila subsp. subsp. maturehydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzymeSEQ ID NO: 3. 125, alanine racemase 1.26E+08 1.00E−155 PseudomonasEmpedobacter AED10581 1.00E−159 126 [Pseudomonas putida brevis putidaGB-1] GB-1 mature gi|126314851|gb|EAZ66019.1| peptide alanine racemasesynthesizing [Pseudomonas enzyme putida GB-1] SEQ ID NO: 3. 127,glutamate 27380813 3.00E−26 Bradyrhizobium Photorhabdus ABM686377.00E−18 128 racemase japonicum luminescens [Bradyrhizobium USDA proteinjaponicum USDA 110 sequence 110] #59. 129, proline racemase, 1.19E+081.00E−103 Stappia Prokaryotic ABU21813 1.00E−101 130 putative [Stappiaaggregata essential aggregata IAM IAM gene 12614] 12614 #34740.gi|118435940|gb|EAV42584.1| proline racemase, putative [Stappiaaggregata IAM 12614] 131, putative proline 1.09E+08 5.00E−75 MyxococcusM. xanthus ABM96637 1.00E−75 132 racemase xanthus protein [Myxococcus DK1622 sequence, xanthus DK 1622] seq id 9726. 133, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−130 134 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.135, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 136 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 137, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−128 138 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 139, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 140 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.141, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 142 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 143, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 144 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 145, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 146 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.147, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 148 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 149, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 150 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 151, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 152 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.153, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 154 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 155, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−133 156 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 157, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 158 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.159, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 160 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 161, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 162 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 163, alanine racemase1.49E+08 1.00E−153 Pseudomonas Empedobacter AED10581 0 164 [Pseudomonasputida F1 brevis putida F1] mature peptide synthesizing enzyme SEQ IDNO: 3. 165, alanine racemase 1.49E+08 1.00E−153 Pseudomonas EmpedobacterAED10581 0 166 [Pseudomonas putida F1 brevis putida F1] mature peptidesynthesizing enzyme SEQ ID NO: 3. 167, proline racemase 887123941.00E−69 Flavobacteriales Bacterial ADS22995 2.00E−57 168[Flavobacteriales bacterium polypeptide bacterium HTCC2170 #10001.HTCC2170] gi|88708932|gb|EAR01166.1| proline racemase [Flavobacterialesbacterium HTCC2170] 169, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 170 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 171, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 172 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.173, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 174 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 175, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 176 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 177, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 178 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.179, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−132 180 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 181, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 182 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 183, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 184 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.185, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 186 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 187, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 188 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 189, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 190 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.191, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 192 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 193, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 194 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 195, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 196 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.197, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 198 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 199, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 200 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 201, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 202 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.203, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 204 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 205, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 206 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 207, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−133 208 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.209, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 210 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 211, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 212 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 213, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−133 214 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.215, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 216 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 217, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 218 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 219, alanine racemase1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 220 [Aeromonassalmonicida brevis salmonicida subsp. mature subsp. salmonicida peptidesalmonicida A449] A449 synthesizing enzyme SEQ ID NO: 3. 221, alanineracemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−128 222[Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 223, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−133 224 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 225, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 226 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 227, alanine racemase 1.18E+08 0Aeromonas Empedobacter AED10581 1.00E−132 228 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 229, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−130 230[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO:3. 231, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 232 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 233, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−128 234 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 235, alanine racemase 1.18E+08 0Aeromonas Empedobacter AED10581 1.00E−128 236 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 237, alanineracemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−128 238[Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 239, alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED105811.00E−128 240 [Aeromonas salmonicida brevis salmonicida subsp. maturesubsp. salmonicida peptide salmonicida A449] A449 synthesizing enzymeSEQ ID NO: 3. 241, alanine racemase 1.45E+08 0 Aeromonas EmpedobacterAED10581 1.00E−130 242 [Aeromonas salmonicida brevis salmonicida subsp.mature subsp. salmonicida peptide salmonicida A449] A449 synthesizingenzyme SEQ ID NO: 3. 243, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−129 244 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 245, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 246 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.247, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 248 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 249, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−129 250 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 251, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−130 252 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.253, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 254 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila putida 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 255, hypothetical 13474743 1.00E−170 MesorhizobiumPropionibacterium ABM37068 1.00E−77 256 protein loti acnes[Mesorhizobium predicted loti]. ORF- encoded polypeptide #300. 257,alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−129258 [Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 259, alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED105811.00E−130 260 [Aeromonas salmonicida brevis salmonicida subsp. maturesubsp. salmonicida peptide salmonicida A449] A449 synthesizing enzymeSEQ ID NO: 3. 261, alanine racemase 1.45E+08 0 Aeromonas EmpedobacterAED10581 1.00E−130 262 [Aeromonas salmonicida brevis salmonicida subsp.mature subsp. salmonicida peptide salmonicida A449] A449 synthesizingenzyme SEQ ID NO: 3. 263, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−128 264 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 265, alanine racemase 1.45E+08 0Aeromonas Empedobacter AED10581 1.00E−129 266 [Aeromonas salmonicidabrevis salmonicida subsp. mature subsp. salmonicida peptide salmonicidaA449] A449 synthesizing enzyme SEQ ID NO: 3. 267, alanine racemase1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 268 [Aeromonassalmonicida brevis salmonicida subsp. mature subsp. salmonicida peptidesalmonicida A449] A449 synthesizing enzyme SEQ ID NO: 3. 269, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 270[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO:3. 271, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−132 272 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 273, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 274 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 275, alanine racemase1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−128 276 [Aeromonassalmonicida brevis salmonicida subsp. mature subsp. salmonicida peptidesalmonicida A449] A449 synthesizing enzyme SEQ ID NO: 3. 277, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−133 278[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO:3. 279, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−132 280 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 281, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 282 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 283, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 284 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.285, alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED105811.00E−130 286 [Aeromonas salmonicida brevis salmonicida subsp. maturesubsp. salmonicida peptide salmonicida A449] A449 synthesizing enzymeSEQ ID NO: 3. 287, alanine racemase 1.45E+08 0 Aeromonas EmpedobacterAED10581 1.00E−129 288 [Aeromonas salmonicida brevis salmonicida subsp.mature subsp. salmonicida peptide salmonicida A449] A449 synthesizingenzyme SEQ ID NO: 3. 289, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−129 290 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 291, alanine racemase 1.18E+08 0Aeromonas Empedobacter AED10581 1.00E−131 292 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 293, alanineracemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−127 294[Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 295, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 296 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 297, proline racemase 1.18E+08 1.00E−104Burkholderia Acinetobacter ADA35228 1.00E−105 298 [Burkholderia phymatumbaumannii phymatum STM815 protein STM815] #19.gi|117982269|gb|EAU96656.1| proline racemase [Burkholderia phymatumSTM815] 299, alanine racemase 1.26E+08 1.00E−170 Pseudomonas T. maritimaAED11803 1.00E−169 300 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 301, alanine racemase 26990430 1.00E−143Pseudomonas T. maritima AED11803 1.00E−144 302 [Pseudomonas putidaD-alanine- putida KT2440] KT2440 D-alanine ligase. 303, hypothetical94414235 4.00E−33 Pseudomonas Pseudomonas ABO82155 4.00E−33 304 proteinaeruginosa aeruginosa PaerP_01003954 PA7 polypeptide [Pseudomonas #3.aeruginosa PA7] 305, AGR_L_3051p 15891641 1.00E−128 Agrobacterium L.pneumophila AEB41596 3.00E−39 306 [Agrobacterium tumefaciens proteintumefaciens]. SEQ ID NO 3367. 307, proline racemase 1.24E+08 3.00E−59Microscilla Bacterial ADS22995 3.00E−47 308 [Microscilla marina marinapolypeptide ATCC 23134] ATCC #10001. gi|123984081|gb|EAY24454.1| 23134proline racemase [Microscilla marina ATCC 23134] 309, alanine racemase1.2E+08 2.00E−57 Stenotrophomonas Prokaryotic ABU41398 8.00E−52 310[Stenotrophomonas maltophilia essential maltophilia R551-3 gene R551-3]#34740. gi|119820021|gb|EAX22642.1| alanine racemase [Stenotrophomonasmaltophilia R551-3] 311, mandelate 83953326 1.00E−143 SulfitobacterKlebsiella ABO61307 2.00E−71 312 racemase/muconate sp. pneumonialactonizing NAS- polypeptide enzyme family 14.1 seqid 7178. protein[Sulfitobacter sp. NAS-14.1] gi|83842294|gb|EAP81462.1| mandelateracemase/muconate lactonizing enzyme family protein [Sulfitobacter sp.NAS-14.1] 313, proline racemase 13473394 1.00E−120 MesorhizobiumBacterial ADS22995 1.00E−120 314 [Mesorhizobium loti polypeptide loti].#10001. 315, proline racemase 88712394 1.00E−106 FlavobacterialesBacterial ADS22995 1.00E−72 316 [Flavobacteriales bacterium polypeptidebacterium HTCC2170 #10001. HTCC2170] gi|88708932|gb|EAR01166.1| prolineracemase [Flavobacteriales bacterium HTCC2170] 317, Mandelate 887125961.00E−110 Flavobacteriales Bacteroides AEX28600 5.00E−34 318racemase/muconate bacterium fragilis lactonizing HTCC2170 strain enzyme14062 [Flavobacteriales protein, bacterium SEQ: 5227. HTCC2170]gi|88709134|gb|EAR01368.1| Mandelate racemase/muconate lactonizingenzyme [Flavobacteriales bacterium HTCC2170] 319, COG3938: Proline84321952 1.00E−60 Pseudomonas Pseudomonas ABO82155 4.00E−61 320 racemaseaeruginosa aeruginosa [Pseudomonas C3719 polypeptide aeruginosa #3.C3719] gi|84328204|ref|ZP_00976211.1| COG3938: Proline racemase[Pseudomonas aeruginosa 2192] gi|107100719|ref|ZP_01364637.1|hypothetical protein PaerPA_01001746 [Pseudomonas aeruginosa PACS2]gi|12616632 321, aspartate 14521575 2.00E−28 Pyrococcus ThermococcusADN46207 7.00E−25 322 racemase abyssi kodakaraensis [Pyrococcus KOD1abyssi]. protein sequence SeqID4. 323, proline racemase, 912173611.00E−152 Psychroflexus Bacterial ADS22995 1.00E−110 324 putativetorquis polypeptide [Psychroflexus ATCC #10001. torquis ATCC 700755700755] gi|91184469|gb|EAS70852.1| proline racemase, putative[Psychroflexus torquis ATCC 700755] 325, proline racemase 1.24E+081.00E−145 Microscilla Bacterial ADS22995 1.00E−126 326 [Microscillamarina marina polypeptide ATCC 23134] ATCC #10001.gi|123984081|gb|EAY24454.1| 23134 proline racemase [Microscilla marinaATCC 23134] 327, proline racemase 1.24E+08 1.00E−147 MicroscillaBacterial ADS22995 1.00E−132 328 [Microscilla marina marina polypeptideATCC 23134] ATCC #10001. gi|123984081|gb|EAY24454.1| 23134 prolineracemase [Microscilla marina ATCC 23134] 329, putative proline 1.09E+081.00E−101 Myxococcus M. xanthus ABM96637 1.00E−101 330 racemase xanthusprotein [Myxococcus DK 1622 sequence, xanthus DK 1622] seq id 9726. 331,AGR_L_3051p 15891641 7.00E−44 Agrobacterium Bacterial ADF07948 5.00E−21332 [Agrobacterium tumefaciens polypeptide tumefaciens]. #19. 333,putative alanine 1.16E+08 9.00E−97 Rhizobium Bacterial ADF07948 6.00E−29334 racemase leguminosarum polypeptide [Rhizobium bv. viciae #19.leguminosarum 3841 bv. viciae 3841] 335, alanine racemase 835909905.00E−78 Moorella Prokaryotic ABU24721 1.00E−63 336 [Moorellathermoacetica essential thermoacetica ATCC gene ATCC 39073] 39073#34740. 337, alanine racemase 83590990 3.00E−77 Moorella ProkaryoticABU24721 1.00E−60 338 [Moorella thermoacetica essential thermoaceticaATCC gene ATCC 39073] 39073 #34740. 339, Alanine racemase 671554691.00E−115 Azotobacter Prokaryotic ABU39795 1.00E−115 340 [Azotobactervinelandii essential vinelandii AvOP] AvOP genegi|67087270|gb|EAM06737.1| #34740. Alanine racemase [Azotobactervinelandii AvOP] 341, mandelate 1.03E+08 2.00E−99 SphingopyxisKlebsiella ABO61307 1.00E−45 342 racemase/muconate alaskensis pneumoniaelactonizing RB2256 polypeptide enzyme seqid 7178. [Sphingopyxisalaskensis RB2256] 343, putative alanine 1.16E+08 1.00E−127 RhizobiumPseudomonas ABO84274 8.00E−42 344 racemase leguminosarum aeruginosa[Rhizobium bv. viciae polypeptide leguminosarum 3841 #3. bv. viciae3841] 345, AGR_L_3051p 15891641 2.00E−79 Agrobacterium PhotorhabdusABM69114 1.00E−27 346 [Agrobacterium tumefaciens luminescenstumefaciens]. protein sequence #59. 347, putative alanine 1.16E+081.00E−114 Rhizobium Prokaryotic ABU41398 1.00E−36 348 racemaseleguminosarum essential [Rhizobium by. viciae gene leguminosarum 3841#34740. bv. viciae 3841] 349, Mandelate 1.49E+08 2.00E−87Methylobacterium Klebsiella ABO61307 1.00E−63 350 racemase/muconate sp.4-46 pneumoniae lactonizing polypeptide enzyme; C- seqid 7178. terminaldomain protein [Methylobacterium sp. 4-46] 351, Mandelate 1.49E+083.00E−93 Sphingomonas Klebsiella ABO61307 3.00E−48 352 racemase/muconatewittichii pneumoniae lactonizing RW1 polypeptide enzyme; C- seqid 7178.terminal domain protein [Sphingomonas wittichii RW1] 353, putativeproline 91779222 1.00E−38 Burkholderia Prokaryotic ABU21813 3.00E−39 354racemase xenovorans essential [Burkholderia LB400 gene xenovorans#34740. LB400] 355 alanine racemase 1.49E+08 2.00E−67 LentisphaeraProkaryotic ABU23921 1.00E−56 356, [Lentisphaera araneosa essentialaraneosa HTCC2155 gene HTCC2155] #34740. 357, alanine racemase 1.18E+081.00E−107 Roseiflexus Prokaryotic ABU23921 5.00E−75 358 [Roseiflexuscastenholzii essential castenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 359, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 1.00E−75 360 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 361, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 5.00E−75 362 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 363, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 3.00E−75 364 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 365, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 3.00E−76 366 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 367, alanine racemase 1.46E+08 1.00E−168 PseudomonasProkaryotic ABU41398 1.00E−160 368 [Pseudomonas mendocina essentialmendocina ymp] ymp gene #34740. 369, diaminopimelate 17232333 3.00E−78Nostoc Bacterial ADS29848 9.00E−79 370 epimerase [Nostoc sp. PCCpolypeptide sp. PCC 7120]. 7120 #10001. 371, diaminopimelate 858579162.00E−76 Syntrophus Bacterial ADN17489 5.00E−60 372 epimeraseaciditrophicus polypeptide [Syntrophus SB #10001. aciditrophicus SB]373, diaminopimelate 17232333 2.00E−71 Nostoc Bacterial ADS298487.00E−72 374 epimerase [Nostoc sp. PCC polypeptide sp. PCC 7120]. 7120#10001. 375, Diaminopimelate 1.26E+08 5.00E−33 Clostridium BacterialADN27169 2.00E−31 376 epimerase thermocellum polypeptide [ClostridiumATCC #10001. thermocellum 27405 ATCC 27405] 377, UDP-N- 1.18E+082.00E−97 Chloroflexus Prokaryotic ABU24769 6.00E−80 378acetylglucosamine aggregans essential 2-epimerase DSM gene [Chloroflexus9485 #34740. aggregans DSM 9485] gi|117997290|gb|EAV11478.1| UDP- N-acetylglucosamine 2-epimerase [Chloroflexus aggregans DSM 9485] 379,NmrA family 1.34E+08 2.00E−88 Burkholderia Bacterial ADS22779 2.00E−84380 protein vietnamiensis polypeptide [Burkholderia G4 #10001.vietnamiensis G4] 381, hypothetical 1.16E+08 2.00E−87 RhizobiumBacterial ADS22779 1.00E−83 382 protein RL1205 leguminosarum polypeptide[Rhizobium bv. viciae #10001. leguminosarum 3841 bv. viciae 3841] 383,similar to 37522668 1.00E−93 Gloeobacter L. pneumophila AEB372824.00E−48 384 chloromuconate violaceus protein cycloisomerase PCC SEQ IDNO [Gloeobacter 7421 3367. violaceus PCC 7421] Geneseq Geneseq DNAPredicted Query Query Subject Subject % % DNA Accession EC DNA ProteinDNA Protein ID ID SEQ ID NO: Description Code Evalue Number LengthLength Length Length Protein DNA 1, 2 Drosophila ABL03829 0.48 2.7.3.687 228 687 228 100 100 melanogaster polypeptide SEQ ID NO 24465. 3, 4Aquifex ABL49607 2.00E−07 5.1.1.1 1017 338 0 338 100 pyrophilus heatresistant alanine racemase encoding DNA. 5, 6 Human AAK80650 0.0322.7.3. 720 239 0 239 80 immune/haematopoietic antigen genomic sequenceSEQ ID NO: 41436. 7, 8 EST clone AAV88076 0.048 1032 343 0 343 89 EP219.9, Pseudomonas ABD08732 1.00E−05 5.1.1.1 1149 382 0 391 78 10 aeruginosapolypeptide #3. 11, Pseudomonas ABD06378 0.013 5.1.1.1 1122 373 0 388 5812 aeruginosa polypeptide #3. 13, Pseudomonas ABD08732 1.00E−08 5.1.1.11065 354 0 353 64 14 aeruginosa polypeptide #3. 15, Klebsiella ACH990540.002 5.1.1.13 702 233 0 231 74 16 pneumoniae polypeptide seqid 7178.17, Human AEB85185 0.009 5.1.1.3 807 268 0 269 72 18 phosphodiesterase4D amino acid sequence N1 SEQ ID NO: 7. 19, M. xanthus ACL64794 0.0375.1.1.3 822 273 0 272 53 20 protein sequence, seq id 9726. 21, BacterialADS61806 1.00E−04 5.2.1.1 768 255 0 265 41 22 polypeptide #10001. 23,Enterobacter AEH55699 0 5.1.1.1 1080 359 0 359 95 24 cloacae proteinamino acid sequence - SEQ ID 5666. 25, Enterobacter AEH55699 0 5.1.1.11080 359 0 359 95 26 cloacae protein amino acid sequence - SEQ ID 5666.27, Prokaryotic ACA27845 0.002 2.7.3. 651 216 0 216 100 28 essentialgene #34740. 29, Pseudomonas ABD15676 1.00E−08 5.1.1.4 1032 343 0 342 6430 aeruginosa polypeptide #3. 31, Pseudomonas ABD15666 9.00E−16 5.1.1.41011 336 0 310 58 32 aeruginosa polypeptide #3. 33, Human AEH98530 0.0135.1.1.1 1116 371 0 385 61 34 cancer associated cDNA SEQ ID NO 9. 35, N.meningitidis AAA81486 3.2 5.1.1. 1101 366 0 368 33 36 partial DNAsequence gnm_640 SEQ ID NO: 640. 37, Pseudomonas ABD12586 0.32 5.1.1.1489 162 0 369 51 38 aeruginosa polypeptide #3. 39, Prokaryotic ACA263328.00E−23 5.1.1.4 372 123 960 318 40 essential gene #34740. 41,Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 97 42 putida racemasepeptide, SEQ ID 5. 43, Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 9644 putida racemase peptide, SEQ ID 5. 45, Pseudomonas ADB99538 0 5.1.1.11230 409 0 409 97 46 putida racemase peptide, SEQ ID 5. 47, PseudomonasADB99538 0 5.1.1.1 1230 409 0 409 97 48 putida racemase peptide, SEQ ID5. 49, Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 96 50 putidaracemase peptide, SEQ ID 5. 51, Pseudomonas ADB99537 1.00E−120 5.1.1.11230 409 0 409 77 52 putida racemase peptide, SEQ ID 5. 53, PseudomonasADB99537 1.00E−124 5.1.1.1 1230 409 0 409 77 54 putida racemase peptide,SEQ ID 5. 55, Pseudomonas ADB99537 1.00E−124 5.1.1.1 1230 409 0 409 7756 putida racemase peptide, SEQ ID 5. 57, Pseudomonas ADB99538 0 5.1.1.11230 409 0 409 96 58 putida racemase peptide, SEQ ID 5. 59, PseudomonasADB99538 0 5.1.1.1 1230 409 0 409 95 60 putida racemase peptide, SEQ ID5. 61, Human ABV52203 0.077 5.1.1.1 1614 537 0 354 19 62 prostateexpression marker cDNA 6604. 63, Pseudomonas ADB99537 0 5.1.1.1 1230 4090 409 98 64 putida racemase peptide, SEQ ID 5. 65, Pseudomonas ADB995370 5.1.1.1 1230 409 0 409 97 66 putida racemase peptide, SEQ ID 5. 67,Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 90 68 putida racemasepeptide, SEQ ID 5. 69, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 9570 putida racemase peptide, SEQ ID 5. 71, Pseudomonas ADB99537 0 5.1.1.11230 409 0 409 96 72 putida racemase peptide, SEQ ID 5. 73, PseudomonasADB99537 0 5.1.1.1 1230 409 0 409 95 74 putida racemase peptide, SEQ ID5. 75, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 95 76 putidaracemase peptide, SEQ ID 5. 77, Pseudomonas ADB99538 0 5.1.1.1 1230 4090 409 95 78 putida racemase peptide, SEQ ID 5. 79, Pseudomonas ADB995380 5.1.1.1 1230 409 0 409 95 80 putida racemase peptide, SEQ ID 5. 81,Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 96 82 putida racemasepeptide, SEQ ID 5. 83, Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 9484 putida racemase peptide, SEQ ID 5. 85, Pseudomonas ADB99537 0 5.1.1.11230 409 0 409 96 86 putida racemase peptide, SEQ ID 5. 87, PseudomonasADB99538 0 5.1.1.1 1230 409 0 409 96 88 putida racemase peptide, SEQ ID5. 89, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 96 90 putidaracemase peptide, SEQ ID 5. 91, Pseudomonas ADB99538 0 5.1.1.1 1230 4090 409 96 92 putida racemase peptide, SEQ ID 5. 93, Pseudomonas ADB995380 5.1.1.1 1230 409 0 409 96 94 putida racemase peptide, SEQ ID 5. 95,Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 95 96 putida racemasepeptide, SEQ ID 5. 97, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 9698 putida racemase peptide, SEQ ID 5. 99, Pseudomonas ADB99537 0 5.1.1.11230 409 0 409 97 100 putida racemase peptide, SEQ ID 5. 101,Enterobacter AEH53103 5.00E−27 5.1.1.1 384 127 1071 356 86 76 102cloacae protein amino acid sequence - SEQ ID 5666. 103, ProkaryoticACA27539 0.13 5.1.1.13 714 237 687 228 32 48 104 essential gene #34740.105, Bovine AEN69487 2.9 5.1.1.4 1002 333 0 333 75 106 ABCG2 related PCRprimer #31. 107, Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 96108 putida racemase peptide, SEQ ID 5. 109, Pseudomonas ADB995386.00E−05 5.1.1.1 1227 408 0 408 95 110 putida racemase peptide, SEQ ID5. 111, Pseudomonas ADB99538 2.00E−11 5.1.1.1 1227 408 0 408 99 112putida racemase peptide, SEQ ID 5. 113, Pseudomonas ADB99538 2.00E−145.1.1.1 1224 407 0 408 92 114 putida racemase peptide, SEQ ID 5. 115,Pseudomonas ADB99538 2.00E−08 5.1.1.1 1227 408 0 408 92 116 putidaracemase peptide, SEQ ID 5. 117, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 92 118 putida racemase peptide, SEQ ID 5. 119,Pseudomonas ADB99538 1.00E−12 5.1.1.1 1227 408 0 408 95 120 putidaracemase peptide, SEQ ID 5. 121, Pseudomonas ADB99538 2.00E−26 5.1.1.11086 361 1227 409 122 putida racemase peptide, SEQ ID 5. 123,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1086 361 0 408 88 124 putidaracemase peptide, SEQ ID 5. 125, Pseudomonas ADB99538 4.00E−95 5.1.1.11086 361 1227 386 126 putida racemase peptide, SEQ ID 5. 127,Prokaryotic ACA37866 2.2 5.1.1.3 261 86 0 265 63 128 essential gene#34740. 129, Prokaryotic ACA26332 5.00E−11 5.1.1.4 957 318 0 310 56 130essential gene #34740. 131, Prokaryotic ACA26332 1.00E−18 5.1.1.4 1074357 960 311 132 essential gene #34740. 133, Pseudomonas ADB995386.00E−11 5.1.1.1 1227 408 0 408 99 134 putida racemase peptide, SEQ ID5. 135, Pseudomonas ADB99538 6.00E−05 5.1.1.1 1227 408 0 408 95 136putida racemase peptide, SEQ ID 5. 137, Pseudomonas ADB99538 0.0045.1.1.1 1221 406 0 408 95 138 putida racemase peptide, SEQ ID 5. 139,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1224 407 0 408 99 140 putidaracemase peptide, SEQ ID 5. 141, Pseudomonas ADB99538 1.00E−15 5.1.1.11227 408 0 408 89 142 putida racemase peptide, SEQ ID 5. 143,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 100 144 putidaracemase peptide, SEQ ID 5. 145, Pseudomonas ADB99538 2.00E−11 5.1.1.11224 407 0 408 99 146 putida racemase peptide, SEQ ID 5. 147,Pseudomonas ADB99538 3.00E−19 5.1.1.1 1224 407 0 408 97 148 putidaracemase peptide, SEQ ID 5. 149, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 93 150 putida racemase peptide, SEQ ID 5. 151,Pseudomonas ADB99538 3.00E−19 5.1.1.1 1227 408 0 408 92 152 putidaracemase peptide, SEQ ID 5. 153, Pseudomonas ADB99538 3.00E−19 5.1.1.11224 407 0 408 97 154 putida racemase peptide, SEQ ID 5. 155,Pseudomonas ADB99538 2.00E−14 5.1.1.1 1227 408 0 408 96 156 putidaracemase peptide, SEQ ID 5. 157, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 92 158 putida racemase peptide, SEQ ID 5. 159,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 92 160 putidaracemase peptide, SEQ ID 5. 161, Pseudomonas ADB99538 3.00E−19 5.1.1.11227 408 0 408 97 162 putida racemase peptide, SEQ ID 5. 163,Pseudomonas ADB99538 1.00E−64 5.1.1.1 1086 361 1227 386 164 putidaracemase peptide, SEQ ID 5. 165, Pseudomonas ADB99538 2.00E−91 5.1.1.11083 360 1227 386 166 putida racemase peptide, SEQ ID 5. 167, BacterialADS60041 0.007 5.1.1.4 657 218 0 335 58 168 polypeptide #10001. 169,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 97 170 putidaracemase peptide, SEQ ID 5. 171, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 96 172 putida racemase peptide, SEQ ID 5. 173,Pseudomonas ADB99538 6.00E−05 5.1.1.1 1224 407 0 408 95 174 putidaracemase peptide, SEQ ID 5. 175, Pseudomonas ADB99538 2.00E−14 5.1.1.11227 408 0 408 89 176 putida racemase peptide, SEQ ID 5. 177,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 99 178 putidaracemase peptide, SEQ ID 5. 179, Pseudomonas ADB99538 3.00E−10 5.1.1.11227 408 0 408 99 180 putida racemase peptide, SEQ ID 5. 181,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 99 182 putidaracemase peptide, SEQ ID 5. 183, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 100 184 putida racemase peptide, SEQ ID 5. 185,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 89 186 putidaracemase peptide, SEQ ID 5. 187, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 99 188 putida racemase peptide, SEQ ID 5. 189,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 99 190 putidaracemase peptide, SEQ ID 5. 191, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 91 192 putida racemase peptide, SEQ ID 5. 193,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 92 194 putidaracemase peptide, SEQ ID 5. 195, Pseudomonas ADB99537 6.00E−11 5.1.1.11227 408 0 408 92 196 putida racemase peptide, SEQ ID 5. 197,Pseudomonas ADB99538 2.00E−14 5.1.1.1 1227 408 0 408 91 198 putidaracemase peptide, SEQ ID 5. 199, Pseudomonas ADB99538 1.00E−15 5.1.1.11227 408 0 408 90 200 putida racemase peptide, SEQ ID 5. 201,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 89 202 putidaracemase peptide, SEQ ID 5. 203, Pseudomonas ADB99538 4.00E−18 5.1.1.11227 408 0 408 92 204 putida racemase peptide, SEQ ID 5. 205,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 89 206 putidaracemase peptide, SEQ ID 5. 207, Pseudomonas ADB99538 3.00E−16 5.1.1.11224 407 0 408 92 208 putida racemase peptide, SEQ ID 5. 209,Pseudomonas ADB99538 1.00E−12 5.1.1.1 1227 408 0 408 91 210 putidaracemase peptide, SEQ ID 5. 211, Pseudomonas ADB99538 2.00E−14 5.1.1.11224 407 0 408 89 212 putida racemase peptide, SEQ ID 5. 213,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1224 407 0 408 91 214 putidaracemase peptide, SEQ ID 5. 215, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 96 216 putida racemase peptide, SEQ ID 5. 217,Pseudomonas ADB99538 7.00E−14 5.1.1.1 1224 407 0 408 92 218 putidaracemase peptide, SEQ ID 5. 219, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 220 putida racemase peptide, SEQ ID 5. 221,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1224 407 0 408 95 222 putidaracemase peptide, SEQ ID 5. 223, Pseudomonas ADB99538 2.00E−14 5.1.1.11224 407 0 408 93 224 putida racemase peptide, SEQ ID 5. 225,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 94 226 putidaracemase peptide, SEQ ID 5. 227, Pseudomonas ADB99538 2.00E−14 5.1.1.11227 408 0 408 91 228 putida racemase peptide, SEQ ID 5. 229,Pseudomonas ADB99538 3.00E−13 5.1.1.1 1227 408 0 408 91 230 putidaracemase peptide, SEQ ID 5. 231, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 232 putida racemase peptide, SEQ ID 5. 233,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 234 putidaracemase peptide, SEQ ID 5. 235, Pseudomonas ADB99538 6.00E−05 5.1.1.11224 407 0 408 95 236 putida racemase peptide, SEQ ID 5. 237,Pseudomonas ADB99538 6.00E−05 5.1.1.1 1227 408 0 408 96 238 putidaracemase peptide, SEQ ID 5. 239, Pseudomonas ADB99538 6.00E−11 5.1.1.11224 407 0 408 94 240 putida racemase peptide, SEQ ID 5. 241,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 242 putidaracemase peptide, SEQ ID 5. 243, Pseudomonas ADB99538 4.00E−06 5.1.1.11227 408 0 408 95 244 putida racemase peptide, SEQ ID 5. 245,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1227 408 0 408 95 246 putidaracemase peptide, SEQ ID 5. 247, Pseudomonas ADB99538 4.00E−06 5.1.1.11227 408 0 408 96 248 putida racemase peptide, SEQ ID 5. 249,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1227 408 0 408 95 250 putidaracemase peptide, SEQ ID 5. 251, Pseudomonas ADB99538 4.00E−06 5.1.1.11227 408 0 408 95 252 putida racemase peptide, SEQ ID 5. 253,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1224 407 0 408 95 254 putidaracemase peptide, SEQ ID 5. 255, Plant ADT17374 0.2 5.1.1.1 1086 3611827 608 83 80 256 polypeptide, SEQ ID 5546. 257, Pseudomonas ADB995382.00E−08 5.1.1.1 1227 408 0 408 95 258 putida racemase peptide, SEQ ID5. 259, Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 260putida racemase peptide, SEQ ID 5. 261, Pseudomonas ADB99538 2.00E−085.1.1.1 1227 408 0 408 94 262 putida racemase peptide, SEQ ID 5. 263,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 264 putidaracemase peptide, SEQ ID 5. 265, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 266 putida racemase peptide, SEQ ID 5. 267,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 94 268 putidaracemase peptide, SEQ ID 5. 269, Pseudomonas ADB99538 1.00E−12 5.1.1.11227 408 0 408 92 270 putida racemase peptide, SEQ ID 5. 271,Pseudomonas ADB99538 3.00E−13 5.1.1.1 1230 409 0 408 92 272 putidaracemase peptide, SEQ ID 5. 273, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 96 274 putida racemase peptide, SEQ ID 5. 275,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 97 276 putidaracemase peptide, SEQ ID 5. 277, Pseudomonas ADB99538 2.00E−14 5.1.1.11227 408 0 408 92 278 putida racemase peptide, SEQ ID 5. 279,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 92 280 putidaracemase peptide, SEQ ID 5. 281, Pseudomonas ADB99538 1.00E−18 5.1.1.11227 408 0 408 92 282 putida racemase peptide, SEQ ID 5. 283,Pseudomonas ADB99538 1.00E−18 5.1.1.1 1227 408 0 408 92 284 putidaracemase peptide, SEQ ID 5. 285, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 286 putida racemase peptide, SEQ ID 5. 287,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 288 putidaracemase peptide, SEQ ID 5. 289, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 290 putida racemase peptide, SEQ ID 5. 291,Pseudomonas ADB99538 2.00E−14 5.1.1.1 1227 408 0 408 92 292 putidaracemase peptide, SEQ ID 5. 293, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 94 294 putida racemase peptide, SEQ ID 5. 295,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1227 408 0 408 95 296 putidaracemase peptide, SEQ ID 5. 297, Prokaryotic ACA19650 9.00E−22 5.1.1.4960 319 945 331 298 essential gene #34740. 299, Pseudomonas ADB995385.00E−95 5.1.1.1 1230 409 0 409 73 300 putida racemase peptide, SEQ ID5. 301, Pseudomonas ADB99538 5.00E−27 5.1.1.1 1239 412 1227 409 302putida racemase peptide, SEQ ID 5. 303, Prokaryotic ACA44058 7.00E−145.1.1.4 375 124 0 314 60 304 essential gene #34740. 305, ProkaryoticACA27267 0.21 5.1.1.1 1125 374 1167 388 64 66 306 essential gene #34740.307, Bacterial ADS60041 2.00E−05 5.1.1.4 429 142 0 333 73 308polypeptide #10001. 309, Prokaryotic ACA43665 3.00E−13 5.1.1.1 408 135 0355 80 310 essential gene #34740. 311, Novel mar AAS46252 0.67 5.1.2.2939 312 0 321 81 312 regulated protein (NIMR) #29. 313, C. botulinumAEF99607 0.65 5.1.1.4 909 302 1002 333 70 62 314 active BoNT/A modifiedopen reading frame, SEQ ID No: 7. 315, Haemophilus ADT05504 0.03 5.1.1.4684 227 0 335 79 316 influenzae (NTHi) protein - SEQ ID 618. 317,Prokaryotic ACA23465 2.9 5.5.1.1 1032 343 0 348 57 318 essential gene#34740. 319, Pseudomonas ABD15666 3.00E−05 5.1.1.4 636 211 486 339 320aeruginosa polypeptide #3. 321, Breast ACN89360 0.49 5.1.1.13 699 232687 228 31 46 322 cancer related marker, seq id 2. 323, BacterialADS60041 0.16 5.1.1.4 885 294 0 336 85 324 polypeptide #10001. 325,Bacterial ADS60041 5.00E−08 5.1.1.4 1005 334 0 333 73 326 polypeptide#10001. 327, Bacterial ADS60041 0.012 5.1.1.4 1002 333 0 333 74 328polypeptide #10001. 329, Prokaryotic ACA23259 1.00E−17 5.1.1.4 957 318 0311 58 330 essential gene #34740. 331, Mycobacterium AAI99682 1.15.1.1.1 420 139 1167 388 61 68 332 tuberculosis strain H37Rv genome SEQID NO 2. 333, Plant full ADX51636 0.67 5.1.1.1 939 312 0 377 58 334length insert polynucleotide seqid 4980. 335, Enterobacter AEH542600.053 5.1.1.1 1143 380 0 373 41 336 cloacae protein amino acidsequence - SEQ ID 5666. 337, Bacillus AAD29866 8.00E−04 5.1.1.1 1107 3680 373 44 338 licheniformis araA gene fragment amplifying PCR primer #1.339, Prokaryotic ACA23293 5.00E−05 5.1.1.1 1068 355 0 418 60 340essential gene #34740. 341, Geranylgeranyl ADM98687 0.046 5.5.1.1 990329 0 335 57 342 pyrophosphate synthase polypeptide #7. 343,Streptomyces ADO51695 0.83 5.1.1.1 1134 377 0 377 62 344 cattleya NRRL8057 orfY protein. 345, Prokaryotic ACA25617 2.2 5.1.1.1 777 258 1167388 57 65 346 essential gene #34740. 347, Plant full ADX51636 0.815.1.1.1 1113 370 0 377 58 348 length insert polynucleotide seqid 4980.349, Prokaryotic ACA42641 0.72 5.1.2.2 993 330 0 326 51 350 essentialgene #34740. 351, Klebsiella ACH94858 5.00E−05 5.5.1.1 1005 334 0 354 52352 pneumoniae polypeptide seqid 7178. 353, Prokaryotic ACA196503.00E−22 5.1.1.4 381 126 945 318 354 essential gene #34740. 355Prokaryotic ACA38093 9.00E−04 5.1.1.1 1155 384 0 360 39 356, essentialgene #34740. 357, Prokaryotic ACA40224 0.88 5.1.1.1 1203 400 0 849 52358 essential gene #34740. 359, T. versicolor AAF26441 0.88 5.1.1.1 1203400 0 849 52 360 pyrF PCR primer SEQ ID 5. 361, T. versicolor AAF264410.88 5.1.1.1 1203 400 0 849 52 362 pyrF PCR primer SEQ ID 5. 363, T.versicolor AAF26441 0.88 5.1.1.1 1203 400 0 849 52 364 pyrF PCR primerSEQ ID 5. 365, T. versicolor AAF26441 0.88 5.1.1.1 1203 400 0 849 52 366pyrF PCR primer SEQ ID 5. 367, Prokaryotic ACA43665 7.00E−32 5.1.1.11074 357 0 362 82 368 essential gene #34740. 369, S. lavendulae ADE102360.16 5.1.1.7 861 286 1413 285 370 mct gene mutagenic PCR primer #2. 371,Bacterial ADT46187 2.00E−06 5.1.1.7 813 270 0 277 57 372 polypeptide#10001. 373, Enviromental AEH47413 0.16 5.1.1.7 873 290 900 285 374isolate hydrolase, SEQ ID NO: 44. 375, Bacterial ADT42245 9.2 5.1.1.7828 275 0 280 29 376 polypeptide #10001. 377, Prokaryotic ACA270416.00E−08 5.1.3.14 1200 399 0 386 50 378 essential gene #34740. 379,Bacterial ADS59825 3.00E−06 1.6.5.3 879 292 0 287 55 380 polypeptide#10001. 381, Bacterial ADS59825 1.00E−08 1.6.5.3 879 292 0 289 55 382polypeptide #10001. 383, Novel ADQ53782 3.1 5.1.2.2 1071 356 0 356 50384 canine microarray- related DNA sequence SeqID10021. Geneseq NRGeneseq Protein Geneseq SEQ_ID Accession NR NR Protein Accession ProteinNO. NR Description Code Evalue Organism Description Code Evalue 1, 2hypothetical 15605814 1.00E−128 Aquifex Prokaryotic ABU25646 3.00E−47protein [Aquifex aeolicus essential aeolicus]. gene #34740. GeneseqGeneseq/ Geneseq/ Geneseq/ Geneseq/ Geneseq DNA Geneseq Predicted QueryQuery NR NR NR NR SEQ_ID DNA Accession DNA EC DNA Protein DNA Protein %ID % ID NO. Description Code Evalue Number Length Length Length LengthProtein DNA 1, 2 Drosophila ABL03829 0.48 2.7.3. 687 228 687 228 100 100melanogaster polypeptide SEQ ID NO 24465. Geneseq NR Geneseq Protein SEQID Accession NR Protein Accession NO: NR Description Code EvalueOrganism Description Code Evalue 3, 4 alanine racemase 15606873 0Aquifex Aquifex ABB06296 1.00E−147 [Aquifex aeolicus]. aeolicuspyrophilus heat resistant alanine racemase encoding DNA. 5, 6hypothetical 29832668 1.00E−101 Streptomyces Prokaryotic ABU194995.00E−37 protein SAV6126 avermitilis essential [Streptomyces MA- geneavermitilis MA- 4680 #34740. 4680] 7, 8 hypothetical 29830835 0Streptomyces Propionibacterium ABM54358 2.00E−53 protein SAV4292avermitilis acnes [Streptomyces MA- predicted avermitilis MA- 4680 ORF-4680] encoded polypeptide #300 9, alanine racemase 21223124 1.00E−166Streptomyces Prokaryotic ABU34223 6.00E−90 10 [Streptomyces coelicoloressential coelicolor A3(2)] A3(2) gene #34740. 11, alanine racemase86748627 1.00E−106 Rhodopseudomonas Pseudomonas ABO84274 4.00E−50 12[Rhodopseudomonas palustris aeruginosa palustris HaA2 polypeptide HaA2]#3. 13, alanine racemase 56477426 1.00E−128 Azoarcus ProkaryoticABU41398 1.00E−110 14 [Azoarcus sp. sp. EbN1 essential EbN1] gene#34740. 15, Aspartate 1.49E+08 4.00E−93 Marinobacter Klebsiella ABO655031.00E−71 16 racemase algicola pneumoniae [Marinobacter DG893 polypeptidealgicola DG893] seqid 7178. 17, glutamate 1.11E+08 1.00E−106 CytophagaProkaryotic ABU25174 2.00E−44 18 racemase hutchinsonii essential[Cytophaga ATCC gene hutchinsonii ATCC 33406 #34740. 33406] 19,glutamate 39998014 1.00E−74 Geobacter M. xanthus ABM90755 2.00E−67 20racemase sulfurreducens protein [Geobacter PCA sequence, sulfurreducensseq id PCA] 9726. 21, Putative 33596748 5.00E−40 Bordetella ThermococcusADN46910 5.00E−27 22 decarboxylase parapertussis kodakaraensis[Bordetella 12822 KOD1 parapertussis protein 12822] sequence SeqID4. 23,alanine racemase 1.46E+08 0 Enterobacter Enterobacter AEH63094 0 24[Enterobacter sp. sp. cloacae 638] 638 protein amino acid sequence - SEQID 5666. 25, alanine racemase 1.46E+08 0 Enterobacter EnterobacterAEH63094 0 26 [Enterobacter sp. sp. cloacae 638] 638 protein amino acidsequence - SEQ ID 5666. 27, hypothetical 71083067 1.00E−121 CandidatusProkaryotic ABU24290 2.00E−31 28 protein Pelagibacter essentialSAR11_0361 ubique gene [Candidatus HTCC1062 #34740. Pelagibacter ubiqueHTCC1062] 29, putative proline 1.49E+08 1.00E−130 BrucellaovisPseudomonas ABO82134 1.00E−127 30 racemase ATCC aeruginosa [Brucellaovis 25840 polypeptide ATCC 25840] #3. 31, proline racemase, 1.19E+081.00E−109 Stappia Acinetobacter ADA35228 1.00E−105 32 putative [Stappiaaggregata baumannii aggregata IAM IAM protein 12614] 12614 #19.gi|118435940|gb|EAV42584.1| proline racemase, putative [Stappiaaggregata IAM 12614] 33, alanine racemase 1.11E+08 1.00E−124Mesorhizobium Prokaryotic ABU38829 3.00E−46 34 [Mesorhizobium sp. BNC1essential sp. BNC1] gene #34740. 35, putative alanine 90418439 6.00E−49Aurantimonas Achromobacter AEH19277 5.00E−47 36 racemase sp.xylosoxidans [Aurantimonas sp. SI85-9A1 DTA SI85-9A1] SEQ ID NOgi|90338111|gb|EAS51762.1| 6. putative alanine racemase [Aurantimonassp. SI85-9A1] 37, Alanine racemase 1.45E+08 1.00E−38 MagnetospirillumAquifex ABB06296 2.00E−20 38 [Magnetospirillum gryphiswaldensepyrophilus gryphiswaldense MSR-1 heat MSR-1] resistant alanine racemaseencoding DNA. 39, putative proline 91779222 2.00E−36 BurkholderiaProkaryotic ABU21813 5.00E−37 40 racemase xenovorans essential[Burkholderia LB400 gene xenovorans #34740. LB400] 41, alanine racemase1.49E+08 0 Pseudomonas T. maritima AED11803 0 42 [Pseudomonas putida F1D-alanine- putida F1] D-alanine ligase. 43, alanine racemase 1.49E+08 0Pseudomonas T. maritima AED11803 0 44 [Pseudomonas putida F1 D-alanine-putida F1] D-alanine ligase. 45, alanine racemase 1.49E+08 0 PseudomonasT. maritima AED11803 0 46 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 47, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 48 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 49, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 50 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 51, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 1.00E−180 52 [Pseudomonas putida D-alanine- putidaGB-1] GB-1 D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanineracemase [Pseudomonas putida GB-1] 53, alanine racemase 1.26E+081.00E−180 Pseudomonas T. maritima AED11803 1.00E−180 54 [Pseudomonasputida D-alanine- putida GB-1] GB-1 D-alaninegi|126314851|gb|EAZ66019.1| ligase. alanine racemase [Pseudomonas putidaGB-1] 55, alanine racemase 1.26E+08 1.00E−180 Pseudomonas T. maritimaAED11803 1.00E−179 56 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 57, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 58 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 59, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 60 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 61, alanine racemase 1.48E+08 7.00E−19 FusobacteriumAquifex ABB06296 1.00E−12 62 [Fusobacterium nucleatum pyrophilusnucleatum subsp. subsp. heat polymorphum polymorphum resistant ATCC10953] ATCC alanine 10953 racemase encoding DNA. 63, alanine racemase1.49E+08 0 Pseudomonas T. maritima AED11803 0 64 [Pseudomonas putida F1D-alanine- putida F1] D-alanine ligase. 65, alanine racemase 1.49E+08 0Pseudomonas T. maritima AED11803 0 66 [Pseudomonas putida F1 D-alanine-putida F1] D-alanine ligase. 67, alanine racemase 1.26E+08 0 PseudomonasT. maritima AED11803 0 68 [Pseudomonas putida D-alanine- putida GB-1]GB-1 D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 69, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 70 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 71, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 72 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase alanine racemase[Pseudomonas putida GB-1] 73, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 74 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase alanine racemase[Pseudomonas putida GB-1] 75, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 76 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 77, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 78 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 79, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 80 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 81, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 82 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 83, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 84 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 85, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 86 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 87, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 88 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 89, alanine racemase 26990430 0 Pseudomonas T.maritima AED11803 0 90 [Pseudomonas putida D-alanine- putida KT2440]KT2440 D-alanine ligase. 91, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 92 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 93, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11804 0 94 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 95, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 96 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 97, alanine racemase 1.26E+08 0 Pseudomonas T.maritima AED11803 0 98 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 99, alanine racemase 1.49E+08 0 Pseudomonas T.maritima AED11803 0 100 [Pseudomonas putida F1 D-alanine- putida F1]D-alanine ligase. 101, alanine racemase 15801412 2.00E−58 EscherichiaProkaryotic ABU45089 2.00E−58 102 2, catabolic coli essential[Escherichia coli O157:H7 gene O157:H7 EDL933 #34740. EDL933]. 103,aspartate 14521575 8.00E−29 Pyrococcus Thermococcus ADN46207 2.00E−25104 racemase abyssi kodakaraensis [Pyrococcus KOD1 abyssi]. proteinsequence SeqID4. 105, proline racemase 1.24E+08 1.00E−150 MicroscillaBacterial ADS22995 1.00E−128 106 [Microscilla marina marina polypeptideATCC 23134] ATCC #10001. gi|123984081|gb|EAY24454.1| 23134 prolineracemase [Microscilla marina ATCC 23134] 107, alanine recemase 1.18E+080 Aeromonas Empedobacter AED10581 1.00E−131 108 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 109, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 110[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3111, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 112 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 113, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 114 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 115, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 116 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.117, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 118 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 119, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 120 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 121, alanine racemase1.49E+08 1.00E−136 Pseudomonas T. maritima AED11803 1.00E−137 122[Pseudomonas putida F1 D-alanine- putida F1] D-alanine ligase. 123,alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−127124 [Aeromonas hydrophila brevis hydrophila subsp. subsp. maturehydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzymeSEQ ID NO: 3. 125, alanine racemase 1.26E+08 1.00E−155 PseudomonasEmpedobacter AED10581 1.00E−159 126 [Pseudomonas putida brevis putidaGB-1] GB-1 mature gi|126314851|gb|EAZ66019.1| peptide alanine racemasesynthesizing [Pseudomonas enzyme putida GB-1] SEQ ID NO: 3. 127,glutamate 27380813 3.00E−26 Bradyrhizobium Photorhabdus ABM686377.00E−18 128 racemase japonicum luminescens [Bradyrhizobium USDA proteinjaponicum USDA 110 sequence 110] #59. 129, proline racemase, 1.19E+081.00E−103 Stappia Prokaryotic ABU21813 1.00E−101 130 putative [Stappiaaggregata essential aggregata IAM IAM gene 12614] 12614 #34740.gi|118435940|gb|EAV42584.1| proline racemase, putative [Stappiaaggregata IAM 12614] 131, putative proline 1.09E+08 5.00E−75 MyxococcusM. xanthus ABM96637 1.00E−75 132 racemase xanthus protein [Myxococcus DK1622 sequence xanthus DK 1622] seq id 9726. 133, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−130 134 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.135, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 136 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 137, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−128 138 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 139, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 140 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.141, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 142 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 143, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 144 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 145, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 146 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.147, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 148 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 149, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 150 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 151, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 152 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.153, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 154 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 155, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−133 156 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 157, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 158 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.159, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 160 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 161, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 162 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 163, alanine racemase1.49E+08 1.00E−153 Pseudomonas Empedobacter AED10581 0 164 [Pseudomonasputida F1 brevis putida F1] mature peptide synthesizing enzyme SEQ IDNO: 3. 165, alanine racemase 1.49E+08 1.00E−153 Pseudomonas EmpedobacterAED10581 0 166 [Pseudomonas putida F1 brevis putida F1] mature peptidesynthesizing enzyme SEQ ID NO: 3. 167, proline racemase 887123941.00E−69 Flavobacteriales Bacterial ADS22995 2.00E−57 168[Flavobacteriales bacterium polypeptide bacterium HTCC2170 #10001.HTCC2170] gi|88708932|gb|EAR01166.1| proline racemase [Flavobacterialesbacterium HTCC2170] 169, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 170 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 171, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 172 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.173, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 174 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 175, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 176 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 177, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 178 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.179, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−132 180 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 181, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 182 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 183, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 184 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.185, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 186 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 187, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 188 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 189, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 190 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.191, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 192 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 193, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 194 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 195, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−131 196 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.197, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 198 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 199, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 200 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 201, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 202 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.203, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 204 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 205, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 206 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 207, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−133 208 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.209, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−131 210 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 211, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−131 212 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 213, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−133 214 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.215, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 216 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 217, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 218 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 219, alanine racemase1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 220 [Aeromonassalmonicida brevis salmonicida subsp. mature subsp. salmonicida peptidesalmonicida A449] A449 synthesizing enzyme SEQ ID NO: 3. 221, alanineracemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−128 222[Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 223, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−133 224 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 225, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 226 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 227, alanine racemase 1.18E+08 0Aeromonas Empedobacter AED10581 1.00E−132 228 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 229, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−130 230[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO:3. 231, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 232 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 233, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−128 234 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 235, alanine racemase 1.18E+08 0Aeromonas Empedobacter AED10581 1.00E−128 236 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 237, alanineracemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−128 238[Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 239, alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED105811.00E−128 240 [Aeromonas salmonicida brevis salmonicida subsp. maturesubsp. salmonicida peptide salmonicida A449] A449 synthesizing enzymeSEQ ID NO: 3. 241, alanine racemase 1.45E+08 0 Aeromonas EmpedobacterAED10581 1.00E−130 242 [Aeromonas salmonicida brevis salmonicida subsp.mature subsp. salmonicida peptide salmonicida A449] A449 synthesizingenzyme SEQ ID NO: 3. 243, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−129 244 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 245, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 246 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.247, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−130 248 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 249, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−129 250 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 251, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−130 252 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.253, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 254 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 255, hypothetical 13474743 1.00E−170 MesorhizobiumPropionibacterium ABM37068 1.00E−77 256 protein loti acnes[Mesorhizobium predicted loti]. ORF- encoded polypeptide #300. 257,alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−129258 [Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 259, alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED105811.00E−130 260 [Aeromonas salmonicida brevis salmonicida subsp. maturesubsp. salmonicida peptide salmonicida A449] A449 synthesizing enzymeSEQ ID NO: 3. 261, alanine racemase 1.45E+08 0 Aeromonas EmpedobacterAED10581 1.00E−130 262 [Aeromonas salmonicida brevis salmonicida subsp.mature subsp. salmonicida peptide salmonicida A449] A449 synthesizingenzyme SEQ ID NO: 3. 263, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−128 264 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 265, alanine racemase 1.45E+08 0Aeromonas Empedobacter AED10581 1.00E−129 266 [Aeromonas salmonicidabrevis salmonicida subsp. mature subsp. salmonicida peptide salmonicidaA449] A449 synthesizing enzyme SEQ ID NO: 3. 267, alanine racemase1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−129 268 [Aeromonassalmonicida brevis salmonicida subsp. mature subsp. salmonicida peptidesalmonicida A449] A449 synthesizing enzyme SEQ ID NO: 3. 269, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 270[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO:3. 271, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−132 272 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 273, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−130 274 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 275, alanine racemase1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−128 276 [Aeromonassalmonicida brevis salmonicida subsp. mature subsp. salmonicida peptidesalmonicida A449] A449 synthesizing enzyme SEQ ID NO: 3. 277, alanineracemase 1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−133 278[Aeromonas hydrophila brevis hydrophila subsp. subsp. mature hydrophilaATCC hydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO:3. 279, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−132 280 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 281, alanine racemase 1.18E+08 0 AeromonasEmpedobacter AED10581 1.00E−132 282 [Aeromonas hydrophila brevishydrophila subsp. subsp. mature hydrophila ATCC hydrophila peptide 7966]ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 283, alanine racemase1.18E+08 0 Aeromonas Empedobacter AED10581 1.00E−132 284 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.285, alanine racemase 1.45E+08 0 Aeromonas Empedobacter AED105811.00E−130 286 [Aeromonas salmonicida brevis salmonicida subsp. maturesubsp. salmonicida peptide salmonicida A449] A449 synthesizing enzymeSEQ ID NO: 3. 287, alanine racemase 1.45E+08 0 Aeromonas EmpedobacterAED10581 1.00E−129 288 [Aeromonas salmonicida brevis salmonicida subsp.mature subsp. salmonicida peptide salmonicida A449] A449 synthesizingenzyme SEQ ID NO: 3. 289, alanine racemase 1.45E+08 0 AeromonasEmpedobacter AED10581 1.00E−129 290 [Aeromonas salmonicida brevissalmonicida subsp. mature subsp. salmonicida peptide salmonicida A449]A449 synthesizing enzyme SEQ ID NO: 3. 291, alanine racemase 1.18E+08 0Aeromonas Empedobacter AED10581 1.00E−131 292 [Aeromonas hydrophilabrevis hydrophila subsp. subsp. mature hydrophila ATCC hydrophilapeptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3. 293, alanineracemase 1.45E+08 0 Aeromonas Empedobacter AED10581 1.00E−127 294[Aeromonas salmonicida brevis salmonicida subsp. mature subsp.salmonicida peptide salmonicida A449] A449 synthesizing enzyme SEQ IDNO: 3. 295, alanine racemase 1.18E+08 0 Aeromonas Empedobacter AED105811.00E−129 296 [Aeromonas hydrophila brevis hydrophila subsp. subsp.mature hydrophila ATCC hydrophila peptide 7966] ATCC synthesizing 7966enzyme SEQ ID NO: 3. 297, proline racemase 1.18E+08 1.00E−104Burkholderia Acinetobacter ADA35228 1.00E−105 298 [Burkholderia phymatumbaumannii phymatum STM815 protein STM815] #19.gi|117982269|gb|EAU96656.1| proline racemase [Burkholderia phymatumSTM815] 299, alanine racemase 1.26E+08 1.00E−170 Pseudomonas T. maritimaAED11803 1.00E−169 300 [Pseudomonas putida D-alanine- putida GB-1] GB-1D-alanine gi|126314851|gb|EAZ66019.1| ligase. alanine racemase[Pseudomonas putida GB-1] 301, alanine racemase 26990430 1.00E−143Pseudomonas T. maritima AED11803 1.00E−144 302 [Pseudomonas putidaD-alanine- putida KT2440] KT2440 D-alanine ligase. 303, hypothetical94414235 4.00E−33 Pseudomonas Pseudomonas ABO82155 4.00E−33 304 proteinaeruginosa aeruginosa PaerP_01003954 PA7 polypeptide [Pseudomonas #3.aeruginosa PA7] 305, AGR_L_3051p 15891641 1.00E−128 Agrobacterium L.pneumophila AEB41596 3.00E−39 306 [Agrobacterium tumefaciens proteintumefaciens]. SEQ ID NO 3367. 307, proline racemase 1.24E+08 3.00E−59Microscilla Bacterial ADS22995 3.00E−47 308 [Microscilla marina marinapolypeptide ATCC 23134] ATCC #10001. gi|123984081|gb|EAY24454.1| 23134proline racemase [Microscilla marina ATCC 23134] 309, alanine racemase1.2E+08 2.00E−57 Stenotrophomonas Prokaryotic ABU41398 8.00E−52 310[Stenotrophomonas maltophilia essential maltophilia R551-3 gene R551-3]#34740. gi|119820021|gb|EAX22642.1| alanine racemase [Stenotrophomonasmaltophilia R551-3] 311, mandelate 83953326 1.00E−143 SulfitobacterKlebsiella ABO61307 2.00E−71 312 racemase/muconate sp. pneumonialactonizing NAS- polypeptide enzyme family 14.1 seqid 7178. protein[Sulfitobacter sp. NAS-14.1] gi|83842294|gb|EAP81462.1| mandelateracemase/muconate lactonizing enzyme family protein [Sulfitobacter sp.NAS-14.1] 313, proline racemase 13473394 1.00E−120 MesorhizobiumBacterial ADS22995 1.00E−120 314 [Mesorhizobium loti polypeptide loti].#10001. 315, proline racemase 88712394 1.00E−106 FlavobacterialesBacterial ADS22995 1.00E−72 316 [Flavobacteriales bacterium polypeptidebacterium HTCC2170 #10001. HTCC2170] gi|88708932|gb|EAR01166.1| prolineracemase [Flavobacteriales bacterium HTCC2170] 317, Mandelate 887125961.00E−110 Flavobacteriales Bacteroides AEX28600 5.00E−34 318racemase/muconate bacterium fragilis lactonizing HTCC2170 strain enzyme14062 [Flavobacteriales protein, bacterium SEQ: 5227. HTCC2170]gi|88709134|gb|EAR01368.1| Mandelate racemase/muconate lactonizingenzyme [Flavobacteriales bacterium HTCC2170] 319, COG3938: Proline84321952 1.00E−60 Pseudomonas Pseudomonas ABO82155 4.00E−61 320 racemaseaeruginosa aeruginosa [Pseudomonas C3719 polypeptide aeruginosa #3.C3719] gi|84328204|ref|ZP_00976211.1| COG3938: Proline racemase[Pseudomonas aeruginosa 2192] gi|107100719|ref|ZP_01364637.1|hypothetical protein PaerPA_01001746 [Pseudomonas aeruginosa PACS2]gi|12616632 321, aspartate 14521575 2.00E−28 Pyrococcus ThermococcusADN46207 7.00E−25 322 racemase abyssi kodakaraensis [Pyrococcus KOD1abyssi]. protein sequence SeqID4. 323, proline racemase, 912173611.00E−152 Psychroflexus Bacterial ADS22995 1.00E−110 324 putativetorquis polypeptide [Psychroflexus ATCC #10001. torquis ATCC 700755700755] gi|91184469|gb|EAS70852.1| proline racemase, putative[Psychroflexus torquis ATCC 700755] 325, proline racemase 1.24E+081.00E−145 Microscilla Bacterial ADS22995 1.00E−126 326 [Microscillamarina marina polypeptide ATCC 23134] ATCC #10001.gi|123984081|gb|EAY24454.1| 23134 proline racemase [Microscilla marinaATCC 23134] 327, proline racemase 1.24E+08 1.00E−147 MicroscillaBacterial ADS22995 1.00E−132 328 [Microscilla marina marina polypeptideATCC 23134] ATCC #10001. gi|123984081|gb|EAY24454.1| 23134 prolineracemase [Microscilla marina ATCC 23134] 329, putative proline 1.09E+081.00E−101 Myxococcus M. xanthus ABM96637 1.00E−101 330 racemase xanthusprotein [Myxococcus DK 1622 sequence, xanthus DK 1622] seq id 9726. 331,AGR_L_3051p 15891641 7.00E−44 Agrobacterium Bacterial ADF07948 5.00E−21332 [Agrobacterium tumefaciens polypeptide tumefaciens]. #19. 333,putative alanine 1.16E+08 9.00E−97 Rhizobium Bacterial ADF07948 6.00E−29334 racemase leguminosarum polypeptide [Rhizobium bv. viciae #19.leguminosarum 3841 bv. viciae 3841] 335, alanine racemase 835909905.00E−78 Moorella Prokaryotic ABU24721 1.00E−63 336 [Moorellathermoacetica essential thermoacetica ATCC gene ATCC 39073] 39073#34740. 337, alanine racemase 83590990 3.00E−77 Moorella ProkaryoticABU24721 1.00E−60 338 [Moorella thermoacetica essential thermoaceticaATCC gene ATCC 39073] 39073 #34740. 339, Alanine racemase 671554691.00E−115 Azotobacter Prokaryotic ABU39795 1.00E−115 340 [Azotobactervinelandii essential vinelandii AvOP] AvOP genegi|67087270|gb|EAM06737.1| #34740. Alanine racemase [Azotobactervinelandii AvOP] 341, mandelate 1.03E+08 2.00E−99 SphingopyxisKlebsiella ABO61307 1.00E−45 342 racemase/muconate alaskensis pneumoniaelactonizing RB2256 polypeptide enzyme seqid 7178. [Sphingopyxisalaskensis RB2256] 343, putative alanine 1.16E+08 1.00E−127 RhizobiumPseudomonas ABO84274 8.00E−42 344 racemase leguminosarum aeruginosa[Rhizobium bv. viciae polypeptide leguminosarum 3841 #3. bv. viciae3841] 345, AGR_L_3051p 15891641 2.00E−79 Agrobacterium PhotorhabdusABM69114 1.00E−27 346 [Agrobacterium tumefaciens luminescenstumefaciens]. protein sequence #59. 347, putative alanine 1.16E+081.00E−114 Rhizobium Prokaryotic ABU41398 1.00E−36 348 racemaseleguminosarum essential [Rhizobium bv. viciae gene leguminosarum 3841#34740. bv. viciae 3841] 349, Mandelate 1.49E+08 2.00E−87Methylobacterium Klebsiella ABO61307 1.00E−63 350 racemase/muconate sp.4-46 pneumoniae lactonizing polypeptide enzyme; C- seqid 7178. terminaldomain protein [Methylobacterium sp. 4-46] 351, Mandelate 1.49E+083.00E−93 Sphingomonas Klebsiella ABO61307 3.00E−48 352 racemase/muconatewittichii pneumoniae lactonizing RW1 polypeptide enzyme; C- seqid 7178.terminal domain protein [Sphingomonas wittichii RW1] 353, putativeproline 91779222 1.00E−38 Burkholderia Prokaryotic ABU21813 3.00E−39 354racemase xenovorans essential [Burkholderia LB400 gene xenovorans#34740. LB400] 355, alanine racemase 1.49E+08 2.00E−67 LentisphaeraProkaryotic ABU23921 1.00E−56 356 [Lentisphaera araneosa essentialaraneosa HTCC2155 gene HTCC2155] #34740. 357, alanine racemase 1.18E+081.00E−107 Roseiflexus Prokaryotic ABU23921 5.00E−75 358 [Roseiflexuscastenholzii essential castenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 359, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 1.00E−75 360 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 361, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 5.00E−75 362 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 363, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 3.00E−75 364 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 365, alanine racemase 1.18E+08 1.00E−107 RoseiflexusProkaryotic ABU23921 3.00E−76 366 [Roseiflexus castenholzii essentialcastenholzii DSM DSM gene 13941] 13941 #34740.gi|118010932|gb|EAV24951.1| alanine racemase [Roseiflexus castenholziiDSM 13941] 367, alanine racemase 1.46E+08 1.00E−168 PseudomonasProkaryotic ABU41398 1.00E−160 368 [Pseudomonas mendocina essentialmendocina ymp] ymp gene #34740. 369, diaminopimelate 17232333 3.00E−78Nostoc Bacterial ADS29848 9.00E−79 370 epimerase [Nostoc sp. PCCpolypeptide sp. PCC 7120]. 7120 #10001. 371, diaminopimelate 858579162.00E−76 Syntrophus Bacterial ADN17489 5.00E−60 372 epimeraseaciditrophicus polypeptide [Syntrophus SB #10001. aciditrophicus SB]373, diaminopimelate 17232333 2.00E−71 Nostoc Bacterial ADS298487.00E−72 374 epimerase [Nostoc sp. PCC polypeptide sp. PCC 7120]. 7120#10001. 375, Diaminopimelate 1.26E+08 5.00E−33 Clostridium BacterialADN27169 2.00E−31 376 epimerase thermocellum polypeptide [ClostridiumATCC #10001. thermocellum 27405 ATCC 27405] 377, UDP-N- 1.18E+082.00E−97 Chloroflexus Prokaryotic ABU24769 6.00E−80 378acetylglucosamine aggregans essential 2-epimerase DSM gene [Chloroflexus9485 #34740. aggregans DSM 9485] gi|117997290|gb|EAV11478.1| UDP- N-acetylglucosamine 2-epimerase [Chloroflexus aggregans DSM 9485] 379,NmrA family 1.34E+08 2.00E−88 Burkholderia Bacterial ADS22779 2.00E−84380 protein vietnamiensis polypeptide [Burkholderia G4 #10001.vietnamiensis G4] 381, hypothetical 1.16E+08 2.00E−87 RhizobiumBacterial ADS22779 1.00E−83 382 protein RL1205 leguminosarum polypeptide[Rhizobium bv. viciae #10001. leguminosarum 3841 bv. viciae 3841] 383,similar to 37522668 1.00E−93 Gloeobacter L. pneumophila AEB372824.00E−48 384 chloromuconate violaceus protein cycloisomerase PCC SEQ IDNO [Gloeobacter 7421 3367. violaceus PCC 7421] 385, AurantimonasAEH19277 5.00E−47 386 sp. SI85-9A1 387, Azoarcus ABU41398 1.00E−110 388sp. EbN1 389, Enterobacter AEH63094 0 390 sp. 638 391, EnterobacterAEH63094 0 392 sp. 638 393, Aquifex ABB06296 1.00E−147 394 aeolicus VF5395, Streptomyces ABU34223 6.00E−90 396 coelicolor A3(2) 397,Rhodopseudomonas ABO84274 4.00E−50 398 palustris HaA2 399, PseudomonasAED11803 0 400 putida F1 401, Pseudomonas AED11803 1.00E−174 402 putidaGB-1 403, Pseudomonas AED11804 1.00E−175 404 putida GB-1 405,Pseudomonas AED11804 0 406 putida F1 407, Pseudomonas AED11803 0 408putida F1 409, Pseudomonas AED11803 0 410 putida F1 411, alpha ABB062969.00E−13 412 proteobacterium HTCC2255 413, Streptomyces ABM543582.00E−53 414 avermitilis MA- 4680 415, Aeromonas AED10581 1.00E−129 416hydrophila subsp. hydrophila ATCC 7966 417, Aeromonas AED10581 1.00E−131418 hydrophila subsp. hydrophila ATCC 7966 419, Aeromonas AED105811.00E−132 420 hydrophila subsp. hydrophila ATCC 7966 421, AeromonasAED10581 1.00E−131 422 hydrophila subsp. hydrophila ATCC 7966 423,Pseudomonas AED11803 0 424 putida F1 425, Pseudomonas AED11803 1.00E−137426 putida F1 427, Pseudomonas AED11803 1.00E−179 428 putida GB-1 429,Pseudomonas AED11804 0 430 putida F1 431, Pseudomonas AED11803 0 432putida F1 433, Pseudomonas AED11804 1.00E−174 434 putida GB-1 435,Aeromonas AED10581 1.00E−131 436 hydrophila subsp. hydrophila ATCC 7966437, Pyrococcus ADN46207 2.00E−25 438 abyssi GE5 439, Aeromonas AED105811.00E−127 440 hydrophila subsp. hydrophila ATCC 7966 441, AeromonasAED10581 1.00E−133 442 hydrophila subsp. hydrophila ATCC 7966 443,Aeromonas AED10581 1.00E−132 444 hydrophila subsp. hydrophila ATCC 7966445, Aeromonas AED10581 1.00E−128 446 salmonicida subsp. salmonicidaA449 447, Aeromonas AED10581 1.00E−129 448 hydrophila subsp. hydrophilaATCC 7966 449, Aeromonas AED10581 1.00E−130 450 hydrophila subsp.hydrophila ATCC 7966 451, Aeromonas AED10581 1.00E−130 452 hydrophilasubsp. hydrophila ATCC 7966 453, Aeromonas AED10581 1.00E−129 454salmonicida subsp. salmonicida A449 455, Stappia ABU21813 1.00E−101 456aggregata IAM 12614 457, Myxococcus ABM96637 1.00E−75 458 xanthus DK1622 459, Aeromonas AED10581 1.00E−130 460 hydrophila subsp. hydrophilaATCC 7966 461, Pseudomonas AED10581 1.00E−159 462 putida GB-1 463,Mesorhizobium ABU38829 3.00E−46 464 sp. BNC1 465, Microscilla ADS229951.00E−128 466 marina ATCC 23134 467, Burkholderia ADA35228 1.00E−105 468phymatum STM815 469, Sulfitobacter ABO61307 2.00E−71 470 sp. NAS- 14.1471, Pseudomonas ABU41398 1.00E−160 472 mendocina ymp 473, AeromonasAED10581 1.00E−131 474 hydrophila subsp. hydrophila ATCC 7966 475,Pseudomonas AED10581 1.00E−167 476 putida GB-1 477, Pseudomonas AED118031.00E−141 478 putida KT2440 479, protein of 87200831 1.00E−137Novosphingobium Klebsiella ABO64182 1.00E−113 480 unknown functionaromaticivorans pneumoniae DUF453 DSM polypeptide [Novosphingobium 12444seqid 7178. aromaticivorans DSM 12444] 481, alanine racemase 1.18E+081.00E−177 Aeromonas Empedobacter AED10581 1.00E−129 482 [Aeromonashydrophila brevis hydrophila subsp. subsp. mature hydrophila ATCChydrophila peptide 7966] ATCC synthesizing 7966 enzyme SEQ ID NO: 3.483, threonine aldolase 16127336 0 Caulobacter 484 family proteincrescentus [Caulobacter CB15 crescentus CB15] gi|13424764|gb|AAK25068.1|threonine aldolase family protein [Caulobacter crescentus CB15] 485,hypothetical 15891036 0 Agrobacterium 486 protein tumefaciens AGR_L_1837str. [Agrobacterium C58 tumefaciens str. C58]gi|17937630|ref|NP_534419.1| hypothetical protein Atu3927 [Agrobacteriumtumefaciens str. C58] gi|15159365|gb|AAK89493.1| AGR_L_1837p[Agrobacterium tumefaciens str. C58] gi|17742368|gb|AAL44735.1|conserved hypothetical protein [Agrobacterium tumefaciens str. C58] 487,hypothetical 15891021 0 Agrobacterium 488 protein tumefaciens AGR_L_1808str. [Agrobacterium C58 tumefaciens str. C58] gi|15159347|gb|AAK89478.1|AGR_L_1808p [Agrobacterium tumefaciens str. C58] Geneseq Geneseq DNAPredicted Query Query Subject Subject % % DNA Accession EC DNA ProteinDNA Protein ID ID SEQ ID NO: Description Code Evalue Number LengthLength Length Length Protein DNA 3, 4 Aquifex ABL49607 2.00E−07 5.1.1.11017 338 0 338 100 pyrophilus heat resistant alanine racemase encodingDNA. 5, 6 Human AAK80650 0.032 2.7.3. 720 239 0 239 80immune/haematopoietic antigen genomic sequence SEQ ID NO: 41436. 7, 8EST clone AAV88076 0.048 1032 343 0 343 89 EP219. 9, PseudomonasABD08732 1.00E−05 5.1.1.1 1149 382 0 391 78 10 aeruginosa polypeptide#3. 11, Pseudomonas ABD06378 0.013 5.1.1.1 1122 373 0 388 58 12aeruginosa polypeptide #3. 13, Pseudomonas ABD08732 1.00E−08 5.1.1.11065 354 0 353 64 14 aeruginosa polypeptide #3. 15, Klebsiella ACH990540.002 5.1.1.13 702 233 0 231 74 16 pneumoniae polypeptide seqid 7178.17, Human AEB85185 0.009 5.1.1.3 807 268 0 269 72 18 phosphodiesterase4D amino acid sequence N1 SEQ ID NO: 7. 19, M. xanthus ACL64794 0.0375.1.1.3 822 273 0 272 53 20 protein sequence, seq id 9726. 21, BacterialADS61806 1.00E−04 5.2.1.1 768 255 0 265 41 22 polypeptide #10001. 23,Enterobacter AEH55699 0 5.1.1.1 1080 359 0 359 95 24 cloacae proteinamino acid sequence - SEQ ID 5666. 25, Enterobacter AEH55699 0 5.1.1.11080 359 0 359 95 26 cloacae protein amino acid sequence - SEQ ID 5666.27, Prokaryotic ACA27845 0.002 2.7.3. 651 216 0 216 100 28 essentialgene #34740. 29, Pseudomonas ABD15676 1.00E−08 5.1.1.4 1032 343 0 342 6430 aeruginosa polypeptide #3. 31, Pseudomonas ABD15666 9.00E−16 5.1.1.41011 336 0 310 58 32 aeruginosa polypeptide #3. 33, Human AEH98530 0.0135.1.1.1 1116 371 0 385 61 34 cancer associated cDNA SEQ ID NO 9. 35, N.meningitidis AAA81486 3.2 5.1.1. 1101 366 0 368 33 36 partial DNAsequence gnm_640 SEQ ID NO: 640. 37, Pseudomonas ABD12586 0.32 5.1.1.1489 162 0 369 51 38 aeruginosa polypeptide #3. 39, Prokaryotic ACA263328.00E−23 5.1.1.4 372 123 960 318 40 essential gene #34740. 41,Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 97 42 putida racemasepeptide, SEQ ID 5. 43, Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 9644 putida racemase peptide, SEQ ID 5. 45, Pseudomonas ADB99538 0 5.1.1.11230 409 0 409 97 46 putida racemase peptide, SEQ ID 5. 47, PseudomonasADB99538 0 5.1.1.1 1230 409 0 409 97 48 putida racemase peptide, SEQ ID5. 49, Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 96 50 putidaracemase peptide, SEQ ID 5. 51, Pseudomonas ADB99537 1.00E−120 5.1.1.11230 409 0 409 77 52 putida racemase peptide, SEQ ID 5. 53, PseudomonasADB99537 1.00E−124 5.1.1.1 1230 409 0 409 77 54 putida racemase peptide,SEQ ID 5. 55, Pseudomonas ADB99537 1.00E−124 5.1.1.1 1230 409 0 409 7756 putida racemase peptide, SEQ ID 5. 57, Pseudomonas ADB99538 0 5.1.1.11230 409 0 409 96 58 putida racemase peptide, SEQ ID 5. 59, PseudomonasADB99538 0 5.1.1.1 1230 409 0 409 95 60 putida racemase peptide, SEQ ID5. 61, Human ABV52203 0.077 5.1.1.1 1614 537 0 354 19 62 prostateexpression marker cDNA 6604. 63, Pseudomonas ADB99537 0 5.1.1.1 1230 4090 409 98 64 putida racemase peptide, SEQ ID 5. 65, Pseudomonas ADB995370 5.1.1.1 1230 409 0 409 97 66 putida racemase peptide, SEQ ID 5. 67,Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 90 68 putida racemasepeptide, SEQ ID 5. 69, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 9570 putida racemase peptide, SEQ ID 5. 71, Pseudomonas ADB99537 0 5.1.1.11230 409 0 409 96 72 putida racemase peptide, SEQ ID 5. 73, PseudomonasADB99537 0 5.1.1.1 1230 409 0 409 95 74 putida racemase peptide, SEQ ID5. 75, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 95 76 putidaracemase peptide, SEQ ID 5. 77, Pseudomonas ADB99538 0 5.1.1.1 1230 4090 409 95 78 putida racemase peptida, SEQ ID 5. 79, Pseudomonas ADB995380 5.1.1.1 1230 409 0 409 95 80 putida racemase peptide, SEQ ID 5. 81,Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 96 82 putida racemasepeptide, SEQ ID 5. 83, Pseudomonas ADB99538 0 5.1.1.1 1230 409 0 409 9484 putida racemase peptide, SEQ ID 5. 85, Pseudomonas ADB99537 0 5.1.1.11230 409 0 409 96 86 putida racemase peptide, SEQ ID 5. 87, PseudomonasADB99538 0 5.1.1.1 1230 409 0 409 96 88 putida racemase peptide, SEQ ID5. 89, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 96 90 putidaracemase peptide, SEQ ID 5. 91, Pseudomonas ADB99538 0 5.1.1.1 1230 4090 409 96 92 putida racemase peptide, SEQ ID 5. 93, Pseudomonas ADB995380 5.1.1.1 1230 409 0 409 96 94 putida racemase peptide, SEQ ID 5. 95,Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 95 96 putida racemasepeptide, SEQ ID 5. 97, Pseudomonas ADB99537 0 5.1.1.1 1230 409 0 409 9698 putida racemase peptide, SEQ ID 5. 99, Pseudomonas ADB99537 0 5.1.1.11230 409 0 409 97 100 putida racemase peptide, SEQ ID 5. 101,Enterobacter AEH53103 5.00E−27 5.1.1.1 384 127 1071 356 86 76 102cloacae protein amino acid sequence- SEQ ID 5666. 103, ProkaryoticACA27539 0.13 5.1.1.13 714 237 687 228 32 48 104 essential gene #34740.105, Bovine AEN69487 2.9 5.1.1.4 1002 333 0 333 75 106 ABCG2 related PCRprimer #31. 107, Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 96108 putida racemase peptide, SEQ ID 5. 109, Pseudomonas ADB995386.00E−05 5.1.1.1 1227 408 0 408 95 110 putida racemase peptide, SEQ ID5. 111, Pseudomonas ADB99538 2.00E−11 5.1.1.1 1227 408 0 408 99 112putida racemase peptide, SEQ ID 5. 113, Pseudomonas ADB99538 2.00E−145.1.1.1 1224 407 0 408 92 114 putida racemase peptide, SEQ ID 5. 115,Pseudomonas ADB99538 2.00E−08 5.1.1.1 1227 408 0 408 92 116 putidaracemase peptide, SEQ ID 5. 117, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 92 118 putida racemase peptide, SEQ ID 5. 119,Pseudomonas ADB99538 1.00E−12 5.1.1.1 1227 408 0 408 95 120 putidaracemase peptide, SEQ ID 5. 121, Pseudomonas ADB99538 2.00E−26 5.1.1.11086 361 1227 409 122 putida racemase peptide, SEQ ID 5. 123,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1086 361 0 408 88 124 putidaracemase peptide, SEQ ID 5. 125, Pseudomonas ADB99538 4.00E−95 5.1.1.11086 361 1227 386 126 putida racemase peptide, SEQ ID 5. 127,Prokaryotic ACA37866 2.2 5.1.1.3 261 86 0 265 63 128 essential gene#34740. 129, Prokaryotic ACA26332 5.00E−11 5.1.1.4 957 318 0 310 56 130essential gene #34740. 131, Prokaryotic ACA26332 1.00E−18 5.1.1.4 1074357 960 311 132 essential gene #34740. 133, Pseudomonas ADB995386.00E−11 5.1.1.1 1227 408 0 408 99 134 putida racemase peptide, SEQ ID5. 135, Pseudomonas ADB99538 6.00E−05 5.1.1.1 1227 408 0 408 95 136putida racemase peptide, SEQ ID 5. 137, Pseudomonas ADB99538 0.0045.1.1.1 1221 406 0 408 95 138 putida racemase peptide, SEQ ID 5. 139,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1224 407 0 408 99 140 putidaracemase peptide, SEQ ID 5. 141, Pseudomonas ADB99538 1.00E−15 5.1.1.11227 408 0 408 89 142 putida racemase peptide, SEQ ID 5. 143,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 100 144 putidaracemase peptide, SEQ ID 5. 145, Pseudomonas ADB99538 2.00E−11 5.1.1.11224 407 0 408 99 146 putida racemase peptide, SEQ ID 5. 147,Pseudomonas ADB99538 3.00E−19 5.1.1.1 1224 407 0 408 97 148 putidaracemase peptide, SEQ ID 5. 149, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 93 150 putida racemase peptide, SEQ ID 5. 151,Pseudomonas ADB99538 3.00E−19 5.1.1.1 1227 408 0 408 92 152 putidaracemase peptide, SEQ ID 5. 153, Pseudomonas ADB99538 3.00E−19 5.1.1.11224 407 0 408 97 154 putida racemase peptide, SEQ ID 5. 155,Pseudomonas ADB99538 2.00E−14 5.1.1.1 1227 408 0 408 96 156 putidaracemase peptide, SEQ ID 5. 157, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 92 158 putida racemase peptide, SEQ ID 5. 159,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 92 160 putidaracemase peptide, SEQ ID 5. 161, Pseudomonas ADB99538 3.00E−19 5.1.1.11227 408 0 408 97 162 putida racemase peptide, SEQ ID 5. 163,Pseudomonas ADB99538 1.00E−64 5.1.1.1 1086 361 1227 386 164 putidaracemase peptide, SEQ ID 5. 165, Pseudomonas ADB99538 2.00E−91 5.1.1.11083 360 1227 386 166 putida racemase peptide, SEQ ID 5. 167, BacterialADS60041 0.007 5.1.1.4 657 218 0 335 58 168 polypeptide #10001. 169,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 97 170 putidaracemase peptide, SEQ ID 5. 171, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 96 172 putida racemase peptide, SEQ ID 5. 173,Pseudomonas ADB99538 6.00E−05 5.1.1.1 1224 407 0 408 95 174 putidaracemase peptide, SEQ ID 5. 175, Pseudomonas ADB99538 2.00E−14 5.1.1.11227 408 0 408 89 176 putida racemase peptide, SEQ ID 5. 177,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 99 178 putidaracemase peptide, SEQ ID 5. 179, Pseudomonas ADB99538 3.00E−10 5.1.1.11227 408 0 408 99 180 putida racemase peptide, SEQ ID 5. 181,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 99 182 putidaracemase peptide, SEQ ID 5. 183, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 100 184 putida racemase peptide, SEQ ID 5. 185,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 89 186 putidaracemase peptide, SEQ ID 5. 187, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 99 188 putida racemase peptide, SEQ ID 5. 189,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 99 190 putidaracemase peptide, SEQ ID 5. 191, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 91 192 putida racemase peptide, SEQ ID 5. 193,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 92 194 putidaracemase peptide, SEQ ID 5. 195, Pseudomonas ADB99537 6.00E−11 5.1.1.11227 408 0 408 92 196 putida racemase peptide, SEQ ID 5. 197,Pseudomonas ADB99538 2.00E−14 5.1.1.1 1227 408 0 408 91 198 putidaracemase peptide, SEQ ID 5. 199, Pseudomonas ADB99538 1.00E−15 5.1.1.11227 408 0 408 90 200 putida racemase peptide, SEQ ID 5. 201,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 89 202 putidaracemase peptide, SEQ ID 5. 203, Pseudomonas ADB99538 4.00E−18 5.1.1.11227 408 0 408 92 204 putida racemase peptide, SEQ ID 5. 205,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 89 206 putidaracemase peptide, SEQ ID 5. 207, Pseudomonas ADB99538 3.00E−16 5.1.1.11224 407 0 408 92 208 putida racemase peptide, SEQ ID 5. 209,Pseudomonas ADB99538 1.00E−12 5.1.1.1 1227 408 0 408 91 210 putidaracemase peptide, SEQ ID 5. 211, Pseudomonas ADB99538 2.00E−14 5.1.1.11224 407 0 408 89 212 putida racemase peptide, SEQ ID 5. 213,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1224 407 0 408 91 214 putidaracemase peptide, SEQ ID 5. 215, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 96 216 putida racemase peptide, SEQ ID 5. 217,Pseudomonas ADB99538 7.00E−14 5.1.1.1 1224 407 0 408 92 218 putidaracemase peptide, SEQ ID 5. 219, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 220 putida racemase peptide, SEQ ID 5. 221,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1224 407 0 408 95 222 putidaracemase peptide, SEQ ID 5. 223, Pseudomonas ADB99538 2.00E−14 5.1.1.11224 407 0 408 93 224 putida racemase peptide, SEQ ID 5. 225,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 94 226 putidaracemase peptide, SEQ ID 5. 227, Pseudomonas ADB99538 2.00E−14 5.1.1.11227 408 0 408 91 228 putida racemase peptide, SEQ ID 5. 229,Pseudomonas ADB99538 3.00E−13 5.1.1.1 1227 408 0 408 91 230 putidaracemase peptide, SEQ ID 5. 231, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 232 putida racemase peptide, SEQ ID 5. 233,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 234 putidaracemase peptide, SEQ ID 5. 235, Pseudomonas ADB99538 6.00E−05 5.1.1.11224 407 0 408 95 236 putida racemase peptide, SEQ ID 5. 237,Pseudomonas ADB99538 6.00E−05 5.1.1.1 1227 408 0 408 96 238 putidaracemase peptide, SEQ ID 5. 239, Pseudomonas ADB99538 6.00E−11 5.1.1.11224 407 0 408 94 240 putida racemase peptide, SEQ ID 5. 241,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 242 putidaracemase peptide, SEQ ID 5. 243, Pseudomonas ADB99538 4.00E−06 5.1.1.11227 408 0 408 95 244 putida racemase peptide, SEQ ID 5. 245,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1227 408 0 408 95 246 putidaracemase peptide, SEQ ID 5. 247, Pseudomonas ADB99538 4.00E−06 5.1.1.11227 408 0 408 96 248 putida racemase peptide, SEQ ID 5. 249,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1227 408 0 408 95 250 putidaracemase peptide, SEQ ID 5. 251, Pseudomonas ADB99538 4.00E−06 5.1.1.11227 408 0 408 95 252 putida racemase peptide, SEQ ID 5. 253,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1224 407 0 408 95 254 putidaracemase peptide, SEQ ID 5. 255, Plant ADT17374 0.2 5.1.1.1 1086 3611827 608 83 80 256 polypeptide, SEQ ID 5546. 257, Pseudomonas ADB995382.00E−08 5.1.1.1 1227 408 0 408 95 258 putida racemase peptide, SEQ ID5. 259, Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 260putida racemase peptide, SEQ ID 5. 261, Pseudomonas ADB99538 2.00E−085.1.1.1 1227 408 0 408 94 262 putida racemase peptide, SEQ ID 5. 263,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 264 putidaracemase peptide, SEQ ID 5. 265, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 266 putida racemase peptide, SEQ ID 5. 267,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 94 268 putidaracemase peptide, SEQ ID 5. 269, Pseudomonas ADB99538 1.00E−12 5.1.1.11227 408 0 408 92 270 putida racemase peptide, SEQ ID 5. 271,Pseudomonas ADB99538 3.00E−13 5.1.1.1 1230 409 0 408 92 272 putidaracemase peptide, SEQ ID 5. 273, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 96 274 putida racemase peptide, SEQ ID 5. 275,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 97 276 putidaracemase peptide, SEQ ID 5. 277, Pseudomonas ADB99538 2.00E−14 5.1.1.11227 408 0 408 92 278 putida racemase peptide, SEQ ID 5. 279,Pseudomonas ADB99538 1.00E−15 5.1.1.1 1227 408 0 408 92 280 putidaracemase peptide, SEQ ID 5. 281, Pseudomonas ADB99538 1.00E−18 5.1.1.11227 408 0 408 92 282 putida racemase peptide, SEQ ID 5. 283,Pseudomonas ADB99538 1.00E−18 5.1.1.1 1227 408 0 408 92 284 putidaracemase peptide, SEQ ID 5. 285, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 286 putida racemase peptide, SEQ ID 5. 287,Pseudomonas ADB99538 6.00E−11 5.1.1.1 1227 408 0 408 95 288 putidaracemase peptide, SEQ ID 5. 289, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 95 290 putida racemase peptide, SEQ ID 5. 291,Pseudomonas ADB99538 2.00E−14 5.1.1.1 1227 408 0 408 92 292 putidaracemase peptide, SEQ ID 5. 293, Pseudomonas ADB99538 6.00E−11 5.1.1.11227 408 0 408 94 294 putida racemase peptide, SEQ ID 5. 295,Pseudomonas ADB99538 4.00E−06 5.1.1.1 1227 408 0 408 95 296 putidaracemase peptide, SEQ ID 5. 297, Prokaryotic ACA19650 9.00E−22 5.1.1.4960 319 945 331 298 essential gene #34740. 299, Pseudomonas ADB995385.00E−95 5.1.1.1 1230 409 0 409 73 300 putida racemase peptide, SEQ ID5. 301, Pseudomonas ADB99538 5.00E−27 5.1.1.1 1239 412 1227 409 302putida racemase peptide, SEQ ID 5. 303, Prokaryotic ACA44058 7.00E−145.1.1.4 375 124 0 314 60 304 essential gene #34740. 305, ProkaryoticACA27267 0.21 5.1.1.1 1125 374 1167 388 64 66 306 essential gene #34740.307, Bacterial ADS60041 2.00E−05 5.1.1.4 429 142 0 333 73 308polypeptide #10001. 309, Prokaryotic ACA43665 3.00E−13 5.1.1.1 408 135 0355 80 310 essential gene #34740. 311, Novel mar AAS46252 0.67 5.1.2.2939 312 0 321 81 312 regulated protein (NIMR) #29. 313, C. botulinumAEF99607 0.65 5.1.1.4 909 302 1002 333 70 62 314 active BoNT/A modifiedopen reading frame, SEQ ID No: 7. 315, Haemophilus ADT05504 0.03 5.1.1.4684 227 0 335 79 316 influenzae (NTHi) protein- SEQ ID 618. 317,Prokaryotic ACA23465 2.9 5.5.1.1 1032 343 0 348 57 318 essential gene#34740. 319, Pseudomonas ABD15666 3.00E−05 5.1.1.4 636 211 486 339 320aeruginosa polypeptide #3. 321, Breast ACN89360 0.49 5.1.1.13 699 232687 228 31 46 322 cancer related marker, seq id 2. 323, BacterialADS60041 0.16 5.1.1.4 885 294 0 336 85 324 polypeptide #10001. 325,Bacterial ADS60041 5.00E−08 5.1.1.4 1005 334 0 333 73 326 polypeptide#10001. 327, Bacterial ADS60041 0.012 5.1.1.4 1002 333 0 333 74 328polypeptide #10001. 329, Prokaryotic ACA23259 1.00E−17 5.1.1.4 957 318 0311 58 330 essential gene #34740. 331, Mycobacterium AAI99682 1.15.1.1.1 420 139 1167 388 61 68 332 tuberculosis strain H37Rv genome SEQID NO 2. 333, Plant full ADX51636 0.67 5.1.1.1 939 312 0 377 58 334length insert polynucleotide seqid 4980. 335, Enterobacter AEH542600.053 5.1.1.1 1143 380 0 373 41 336 cloacae protein amino acidsequence - SEQ ID 5666. 337, Bacillus AAD29866 8.00E−04 5.1.1.1 1107 3680 373 44 338 licheniformis araA gene fragment amplifying PCR primer #1.339, Prokaryotic ACA23293 5.00E−05 5.1.1.1 1068 355 0 418 60 340essential gene #34740. 341, Geranylgeranyl ADM98687 0.046 5.5.1.1 909329 0 335 57 342 pyrophosphate synthase polypeptide #7. 343,Streptomyces ADO51695 0.83 5.1.1.1 1134 377 0 377 62 344 cattleya NRRL8057 orfY protein. 345, Prokaryotic ACA25617 2.2 5.1.1.1 777 258 1167388 57 65 346 essential gene #34740. 347, Plant full ADX51636 0.815.1.1.1 1113 370 0 377 58 348 length insert polynucleotide seqid 4980.349, Prokaryotic ACA42641 0.72 5.1.2.2 993 330 0 326 51 350 essentialgene #34740. 351, Klebsiella ACH94858 5.00E−05 5.5.1.1 1005 334 0 354 52352 pneumoniae polypeptide seqid 7178. 353, Prokaryotic ACA196503.00E−22 5.1.1.4 381 126 945 318 354 essential gene #34740. 355,Prokaryotic ACA38093 9.00E−04 5.1.1.1 1155 384 0 360 39 356 essentialgene #34740. 357, Prokaryotic ACA40224 0.88 5.1.1.1 1203 400 0 849 52358 essential gene #34740. 359, T. versicolor AAF26441 0.88 5.1.1.1 1203400 0 849 52 360 pyrF PCR primer SEQ ID 5. 361, T. versicolor AAF264410.88 5.1.1.1 1203 400 0 849 52 362 pyrF PCR primer SEQ ID 5. 363, T.versicolor AAF26441 0.88 5.1.1.1 1203 400 0 849 52 364 pyrF PCR primerSEQ ID 5. 365, T. versicolor AAF26441 0.88 5.1.1.1 1203 400 0 849 52 366pyrF PCR primer SEQ ID 5. 367, Prokaryotic ACA43665 7.00E−32 5.1.1.11074 357 0 362 82 368 essential gene #34740. 369, S. lavendulae ADE102360.16 5.1.1.7 861 286 1413 285 370 mct gene mutagenic PCR primer #2. 371,Bacterial ADT46187 2.00E−06 5.1.1.7 813 270 0 277 57 372 polypeptide#10001. 373, Environmental AEH47413 0.16 5.1.1.7 873 290 900 285 374isolate hydrolase, SEQ ID NO: 44. 375, Bacterial ADT42245 9.2 5.1.1.7828 275 0 280 29 376 polypeptide #10001. 377, Prokaryotic ACA270416.00E−08 5.1.3.14 1200 399 0 386 50 378 essential gene #34740. 379,Bacterial ADS59825 3.00E−06 1.6.5.3 879 292 0 287 55 380 polypeptide#10001. 381, Bacterial ADS59825 1.00E−08 1.6.5.3 879 292 0 289 55 382polypeptide #10001. 383, Novel ADQ53782 3.1 5.1.2.2 1071 356 0 356 50384 canine microarray- related DNA sequence SeqID100 21. 385, 3.45.1.1.— 1101 366 28730 379 34 95 386 387, 1.00E−08 5.1.1.1 1065 354 1464357 57 88 388 389, 0 5.1.1.1 1080 359 1089 362 96 87 390 391, 0 5.1.1.11080 359 1089 362 96 87 392 393, 2.00E−07 5.1.1.1 1017 338 1468 341 7295 394 395, 2.00E−05 5.1.1.1 1149 382 1464 391 50 92 396 397, 0.0145.1.1.1 1122 373 2046 368 38 100 398 399, 0 5.1.1.1 1161 386 1227 409 9692 400 401, 1.00E−120 5.1.1.1 1164 387 1227 409 77 85 402 403, 1.00E−1245.1.1.1 1164 387 1227 409 78 85 404 405, 0 5.1.1.1 1161 386 1227 409 9796 406 407, 0 5.1.1.1 1161 386 1227 409 96 92 408 409, 0 5.1.1.1 1161386 1227 409 96 92 410 411, 0.077 5.1.1.1 1521 506 633 341 28 100 412413, 0.052 1032 343 306 346 37 100 414 415, 6.00E−05 5.1.1.1 1167 3881227 386 61 83 416 417, 2.00E−11 5.1.1.1 1167 388 1227 386 60 83 418419, 2.00E−08 5.1.1.1 1167 388 1227 386 62 88 420 421, 7.00E−11 5.1.1.11167 388 1227 386 61 90 422 423, 0 5.1.1.1 1161 386 1227 409 95 92 424425, 2.00E−26 5.1.1.1 1092 363 1227 409 64 81 426 427, 1.00E−124 5.1.1.11230 409 1227 409 76 85 428 429, 0 5.1.1.1 1230 409 1227 409 97 96 430431, 0 5.1.1.1 1161 386 1227 409 95 91 432 433, 1.00E−124 5.1.1.1 1164387 1227 409 78 85 434 435, 2.00E−14 5.1.1.1 1167 388 1227 386 61 91 436437, 0.14 5.1.1.13 714 237 1056 232 31 100 438 439, 6.00E−11 5.1.1.11095 364 1227 386 61 86 440 441, 2.00E−14 5.1.1.1 1167 388 1227 386 6191 442 443, 7.00E−14 5.1.1.1 1167 388 1227 386 61 81 444 445, 7.00E−115.1.1.1 1167 388 1227 386 59 86 446 447, 4.00E−06 5.1.1.1 1167 388 1227386 61 83 448 449, 4.00E−06 5.1.1.1 1167 388 1227 386 61 83 450 451,7.00E−11 5.1.1.1 1167 388 1227 386 62 86 452 453, 7.00E−11 5.1.1.1 1167388 1227 386 61 86 454 455, 5.00E−11 5.1.1.4 957 318 960 318 60 90 456457, 1.00E−18 5.1.1.4 1074 357 960 311 51 88 458 459, 1.00E−12 5.1.1.11167 388 1227 386 61 90 460 461, 4.00E−95 5.1.1.1 1092 363 1227 386 7785 462 463, 0.015 5.1.1.1 1143 380 801 358 38 100 464 465, 3.1 5.1.1.41002 333 84480 333 65 95 466 467, 1.00E−21 5.1.1.4 960 319 945 331 60 91468 469, 0.74 5.1.2.2 939 312 10118 347 50 93 470 471, 6.00E−45 5.1.1.11074 357 1071 357 78 82 472 473, 7.00E−11 5.1.1.1 1167 388 1227 386 6286 474 475, 5.00E−95 5.1.1.1 1164 387 1227 386 76 85 476 477, 5.00E−275.1.1.1 1176 391 1227 409 63 81 478 479, Bacterial ADS50054 0.79 1056351 0 352 68 480 polypeptide #23667. 481, Pseudomonas ADB99538 6.00E−145.1.1.1 1089 362 0 408 83 482 putida racemase peptide, SEQ ID 5. 483,19172958 0 5.1.1.— 1110 369 4E+06 369 100 100 484 485, 16445346 05.1.1.— 1089 362 2E+06 362 100 100 486 487, 16445346 0 5.1.1.— 1146 3812E+06 397 100 100 488

The invention provides variants of polynucleotides or polypeptides ofthe invention, which comprise sequences modified at one or more basepairs, codons, introns, exons, or amino acid residues (respectively) yetstill retain the biological activity of an isomerase, e.g., a racemase,e.g., an amino acid racemase, an alanine racemase, and/or an epimeraseof the invention. Variants can be produced by any number of meansincluded methods such as, for example, error-prone PCR, shuffling,oligonucleotide-directed mutagenesis, assembly PCR, sexual PCRmutagenesis, in vivo mutagenesis, cassette mutagenesis, recursiveensemble mutagenesis, exponential ensemble mutagenesis, site-specificmutagenesis, gene reassembly (e.g., GeneReassembly, see, e.g., U.S. Pat.No. 6,537,776), GSSM and any combination thereof.

The term “saturation mutagenesis”, “gene site saturation mutagenesis” or“GSSM” includes a method that uses degenerate oligonucleotide primers tointroduce point mutations into a polynucleotide, as described in detail,below.

The term “optimized directed evolution system” or “optimized directedevolution” includes a method for reassembling fragments of relatednucleic acid sequences, e.g., related genes, and explained in detail,below.

The term “synthetic ligation reassembly” or “SLR” includes a method ofligating oligonucleotide fragments in a non-stochastic fashion, andexplained in detail, below.

Generating and Manipulating Nucleic Acids

The invention provides nucleic acids (e.g., nucleic acids encodingpolypeptides having an isomerase activity, e.g., a racemase activity,e.g., an amino acid racemase activity, an alanine racemase activity,and/or an epimerase activity; including enzymes having at least onesequence modification of an exemplary nucleic acid sequence of theinvention (as defined above), wherein the sequence modificationcomprises one or more nucleotide residue changes (or the equivalentthereof), including expression cassettes such as expression vectors,encoding the polypeptides of the invention.

The invention also includes methods for discovering new isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasesequences using the nucleic acids of the invention. The invention alsoincludes methods for inhibiting the expression of isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasegenes, transcripts and polypeptides using the nucleic acids of theinvention. Also provided are methods for modifying the nucleic acids ofthe invention by, e.g., synthetic ligation reassembly, optimizeddirected evolution system and/or saturation mutagenesis.

The nucleic acids of the invention can be made, isolated and/ormanipulated by, e.g., cloning and expression of cDNA libraries,amplification of message or genomic DNA by PCR, and the like.

In one aspect, the invention also provides an isomerase-, e.g., aracemase-, e.g., an amino acid racemase-, an alanine racemase-, and/oran epimerase-isomerase-, e.g., racemase-, e.g., amino acid racemase-,alanine racemase-, and/or epimerase-encoding nucleic acids with a commonnovelty in that they are derived from an environmental source, or abacterial source, or an archaeal source.

In practicing the methods of the invention, homologous genes can bemodified by manipulating a template nucleic acid, as described herein.The invention can be practiced in conjunction with any method orprotocol or device known in the art, which are well described in thescientific and patent literature.

One aspect of the invention is an isolated nucleic acid comprising oneof the sequences of The invention and sequences substantially identicalthereto, the sequences complementary thereto, or a fragment comprisingat least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or500 consecutive bases of one of the sequences of a Sequence of theinvention (or the sequences complementary thereto). The isolated,nucleic acids may comprise DNA, including cDNA, genomic DNA andsynthetic DNA. The DNA may be double-stranded or single-stranded and ifsingle stranded may be the coding strand or non-coding (anti-sense)strand. Alternatively, the isolated nucleic acids may comprise RNA.

Accordingly, another aspect of the invention is an isolated nucleic acidwhich encodes one of the polypeptides of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids of one of the polypeptides of the invention. Thecoding sequences of these nucleic acids may be identical to one of thecoding sequences of one of the nucleic acids of the invention, or afragment thereof or may be different coding sequences which encode oneof the polypeptides of the invention, sequences substantially identicalthereto and fragments having at least 5, 10, 15, 20, 25, 30, 35, 40, 50,75, 100, or 150 consecutive amino acids of one of the polypeptides ofthe invention, as a result of the redundancy or degeneracy of thegenetic code. The genetic code is well known to those of skill in theart and can be obtained, for example, on page 214 of B. Lewin, Genes VI,Oxford University Press, 1997.

The isolated nucleic acid which encodes one of the polypeptides of theinvention and sequences substantially identical thereto, may include,but is not limited to: only the coding sequence of a nucleic acid of theinvention and sequences substantially identical thereto and additionalcoding sequences, such as leader sequences or proprotein sequences andnon-coding sequences, such as introns or non-coding sequences 5′ and/or3′ of the coding sequence. Thus, as used herein, the term“polynucleotide encoding a polypeptide” encompasses a polynucleotidewhich includes only the coding sequence for the polypeptide as well as apolynucleotide which includes additional coding and/or non-codingsequence.

Alternatively, the nucleic acid sequences of the invention and sequencessubstantially identical thereto, may be mutagenized using conventionaltechniques, such as site directed mutagenesis, or other techniquesfamiliar to those skilled in the art, to introduce silent changes intothe polynucleotides of the invention and sequences substantiallyidentical thereto. As used herein, “silent changes” include, forexample, changes which do not alter the amino acid sequence encoded bythe polynucleotide. Such changes may be desirable in order to increasethe level of the polypeptide produced by host cells containing a vectorencoding the polypeptide by introducing codons or codon pairs whichoccur frequently in the host organism.

The invention also relates to polynucleotides which have nucleotidechanges which result in amino acid substitutions, additions, deletions,fusions and truncations in the polypeptides of the invention andsequences substantially identical thereto. Such nucleotide changes maybe introduced using techniques such as site directed mutagenesis, randomchemical mutagenesis, exonuclease III deletion and other recombinant DNAtechniques. Alternatively, such nucleotide changes may be naturallyoccurring allelic variants which are isolated by identifying nucleicacids which specifically hybridize to probes comprising at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutivebases of one of the sequences of The invention and sequencessubstantially identical thereto (or the sequences complementary thereto)under conditions of high, moderate, or low stringency as providedherein.

General Techniques

The nucleic acids used to practice this invention, whether RNA, iRNA,antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybridsthereof, may be isolated from a variety of sources, geneticallyengineered, amplified, and/or expressed/generated recombinantly.Recombinant polypeptides (e.g., isomerases, e.g., racemases, e.g., aminoacid racemases, alanine racemases, and/or epimerases of the invention)generated from these nucleic acids can be individually isolated orcloned and tested for a desired activity. Any recombinant expressionsystem can be used, including bacterial, mammalian, yeast, insect orplant cell expression systems.

Alternatively, these nucleic acids can be synthesized in vitro bywell-known chemical synthesis techniques, as described in, e.g., Adams(1983) J. Am. Chem. Soc. 105:661; Belousov (1997) Nucleic Acids Res.25:3440-3444; Frenkel (1995) Free Radic. Biol. Med. 19:373-380; Blommers(1994) Biochemistry 33:7886-7896; Narang (1979) Meth. Enzymol. 68:90;Brown (1979) Meth. Enzymol. 68:109; Beaucage (1981) Tetra. Lett.22:1859; U.S. Pat. No. 4,458,066.

Techniques for the manipulation of nucleic acids, such as, e.g.,subcloning, labeling probes (e.g., random-primer labeling using Klenowpolymerase, nick translation, amplification), sequencing, hybridizationand the like are well described in the scientific and patent literature,see, e.g., Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2NDED.), Vols. 1-3, Cold Spring Harbor Laboratory, (1989); CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons, Inc.,New York (1997); LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory andNucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y. (1993).

Another useful means of obtaining and manipulating nucleic acids used topractice the methods of the invention is to clone from genomic samples,and, if desired, screen and re-clone inserts isolated or amplified from,e.g., genomic clones or cDNA clones. Sources of nucleic acid used in themethods of the invention include genomic or cDNA libraries contained in,e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos.5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld(1997) Nat. Genet. 15:333-335; yeast artificial chromosomes (YAC);bacterial artificial chromosomes (BAC); P1 artificial chromosomes, see,e.g., Woon (1998) Genomics 50:306-316; P1-derived vectors (PACs), see,e.g., Kern (1997) Biotechniques 23:120-124; cosmids, recombinantviruses, phages or plasmids.

In one aspect, a nucleic acid encoding a polypeptide of the invention isassembled in appropriate phase with a leader sequence capable ofdirecting secretion of the translated polypeptide or fragment thereof.

The invention provides fusion proteins and nucleic acids encoding them.A polypeptide of the invention can be fused to a heterologous peptide orpolypeptide, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification. Peptides and polypeptides of the invention can also besynthesized and expressed as fusion proteins with one or more additionaldomains linked thereto for, e.g., producing a more immunogenic peptide,to more readily isolate a recombinantly synthesized peptide, to identifyand isolate antibodies and antibody-expressing B cells, and the like.Detection and purification facilitating domains include, e.g., metalchelating peptides such as polyhistidine tracts and histidine-tryptophanmodules that allow purification on immobilized metals, protein A domainsthat allow purification on immobilized immunoglobulin, and the domainutilized in the FLAGS extension/affinity purification system (ImmunexCorp, Seattle Wash.). The inclusion of a cleavable linker sequences suchas Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between apurification domain and the motif-comprising peptide or polypeptide tofacilitate purification. For example, an expression vector can includean epitope-encoding nucleic acid sequence linked to six histidineresidues followed by a thioredoxin and an enterokinase cleavage site(see e.g., Williams (1995) Biochemistry 34:1787-1797; Dobeli (1998)Protein Expr. Purif. 12:404-414). The histidine residues facilitatedetection and purification while the enterokinase cleavage site providesa means for purifying the epitope from the remainder of the fusionprotein. Technology pertaining to vectors encoding fusion proteins andapplication of fusion proteins are well described in the scientific andpatent literature, see e.g., Kroll (1993) DNA Cell. Biol., 12:441-53.

The phrases “nucleic acid” or “nucleic acid sequence” as used hereinrefer to an oligonucleotide, nucleotide, polynucleotide, or to afragment of any of these, to DNA or RNA of genomic or synthetic originwhich may be single-stranded or double-stranded and may represent asense or antisense strand, to peptide nucleic acid (PNA), or to anyDNA-like or RNA-like material, natural or synthetic in origin. Thephrases “nucleic acid” or “nucleic acid sequence” includesoligonucleotide, nucleotide, polynucleotide, or to a fragment of any ofthese, to DNA or RNA (e.g., mRNA, rRNA, tRNA, iRNA) of genomic orsynthetic origin which may be single-stranded or double-stranded and mayrepresent a sense or antisense strand, to peptide nucleic acid (PNA), orto any DNA-like or RNA-like material, natural or synthetic in origin,including, e.g., iRNA, ribonucleoproteins (e.g., e.g., double strandediRNAs, e.g., iRNPs). The term encompasses nucleic acids, i.e.,oligonucleotides, containing known analogues of natural nucleotides. Theterm also encompasses nucleic-acid-like structures with syntheticbackbones, see e.g., Mata (1997) Toxicol. Appl. Pharmacol. 144:189-197;Strauss-Soukup (1997) Biochemistry 36:8692-8698; Samstag (1996)Antisense Nucleic Acid Drug Dev 6:153-156. “Oligonucleotide” includeseither a single stranded polydeoxynucleotide or two complementarypolydeoxynucleotide strands that may be chemically synthesized. Suchsynthetic oligonucleotides have no 5′ phosphate and thus will not ligateto another oligonucleotide without adding a phosphate with an ATP in thepresence of a kinase. A synthetic oligonucleotide can ligate to afragment that has not been dephosphorylated.

A “coding sequence of” or a “nucleotide sequence encoding” a particularpolypeptide or protein, is a nucleic acid sequence which is transcribedand translated into a polypeptide or protein when placed under thecontrol of appropriate regulatory sequences.

The term “gene” means the segment of DNA involved in producing apolypeptide chain; it includes regions preceding and following thecoding region (leader and trailer) as well as, where applicable,intervening sequences (introns) between individual coding segments(exons). “Operably linked” as used herein refers to a functionalrelationship between two or more nucleic acid (e.g., DNA) segments.Typically, it refers to the functional relationship of transcriptionalregulatory sequence to a transcribed sequence. For example, a promoteris operably linked to a coding sequence, such as a nucleic acid of theinvention, if it stimulates or modulates the transcription of the codingsequence in an appropriate host cell or other expression system.Generally, promoter transcriptional regulatory sequences that areoperably linked to a transcribed sequence are physically contiguous tothe transcribed sequence, i.e., they are cis-acting. However, sometranscriptional regulatory sequences, such as enhancers, need not bephysically contiguous or located in close proximity to the codingsequences whose transcription they enhance.

The term “expression cassette” as used herein refers to a nucleotidesequence which is capable of affecting expression of a structural gene(i.e., a protein coding sequence, such as an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase of theinvention) in a host compatible with such sequences. Expressioncassettes include at least a promoter operably linked with thepolypeptide coding sequence; and, in one aspect, with other sequences,e.g., transcription termination signals. Additional factors necessary orhelpful in effecting expression may also be used, e.g., enhancers. Thus,expression cassettes also include plasmids, expression vectors,recombinant viruses, any form of recombinant “naked DNA” vector, and thelike. A “vector” comprises a nucleic acid that can infect, transfect,transiently or permanently transduce a cell. It will be recognized thata vector can be a naked nucleic acid, or a nucleic acid complexed withprotein or lipid. The vector in one aspect comprises viral or bacterialnucleic acids and/or proteins, and/or membranes (e.g., a cell membrane,a viral lipid envelope, etc.). Vectors include, but are not limited toreplicons (e.g., RNA replicons, bacteriophages) to which fragments ofDNA may be attached and become replicated. Vectors thus include, but arenot limited to RNA, autonomous self-replicating circular or linear DNAor RNA (e.g., plasmids, viruses, and the like, see, e.g., U.S. Pat. No.5,217,879), and include both the expression and non-expression plasmids.Where a recombinant microorganism or cell culture is described ashosting an “expression vector” this includes both extra-chromosomalcircular and linear DNA and DNA that has been incorporated into the hostchromosome(s). Where a vector is being maintained by a host cell, thevector may either be stably replicated by the cells during mitosis as anautonomous structure, or is incorporated within the host's genome.

As used herein, the term “promoter” includes all sequences capable ofdriving transcription of a coding sequence in a cell, e.g., a plantcell. Thus, promoters used in the constructs of the invention includecis-acting transcriptional control elements and regulatory sequencesthat are involved in regulating or modulating the timing and/or rate oftranscription of a gene. For example, a promoter can be a cis-actingtranscriptional control element, including an enhancer, a promoter, atranscription terminator, an origin of replication, a chromosomalintegration sequence, 5′ and 3′ untranslated regions, or an intronicsequence, which are involved in transcriptional regulation. Thesecis-acting sequences typically interact with proteins or otherbiomolecules to carry out (turn on/off, regulate, modulate, etc.)transcription. “Constitutive” promoters are those that drive expressioncontinuously under most environmental conditions and states ofdevelopment or cell differentiation. “Inducible” or “regulatable”promoters direct expression of the nucleic acid of the invention underthe influence of environmental conditions or developmental conditions.Examples of environmental conditions that may affect transcription byinducible promoters include anaerobic conditions, elevated temperature,drought, or the presence of light.

“Tissue-specific” promoters are transcriptional control elements thatare only active in particular cells or tissues or organs, e.g., inplants or animals. Tissue-specific regulation may be achieved by certainintrinsic factors that ensure that genes encoding proteins specific to agiven tissue are expressed. Such factors are known to exist in mammalsand plants so as to allow for specific tissues to develop.

As used herein, the term “isolated” means that the material (e.g., anucleic acid, a polypeptide, a cell) is removed from its originalenvironment (e.g., the natural environment if it is naturallyoccurring). For example, a naturally-occurring polynucleotide orpolypeptide present in a living animal is not isolated, but the samepolynucleotide or polypeptide, separated from some or all of thecoexisting materials in the natural system, is isolated. Suchpolynucleotides could be part of a vector and/or such polynucleotides orpolypeptides could be part of a composition and still be isolated inthat such vector or composition is not part of its natural environment.As used herein, the term “purified” does not require absolute purity;rather, it is intended as a relative definition. Individual nucleicacids obtained from a library have been conventionally purified toelectrophoretic homogeneity. The sequences obtained from these clonescould not be obtained directly either from the library or from totalhuman DNA. The purified nucleic acids of the invention have beenpurified from the remainder of the genomic DNA in the organism by atleast 10⁴-10⁶ fold. However, the term “purified” also includes nucleicacids that have been purified from the remainder of the genomic DNA orfrom other sequences in a library or other environment by at least oneorder of magnitude, typically two or three orders and more typicallyfour or five orders of magnitude.

As used herein, the term “recombinant” means that the nucleic acid isadjacent to a “backbone” nucleic acid to which it is not adjacent in itsnatural environment. Additionally, to be “enriched” the nucleic acidswill represent 5% or more of the number of nucleic acid inserts in apopulation of nucleic acid backbone molecules. Backbone moleculesaccording to the invention include nucleic acids such as expressionvectors, self-replicating nucleic acids, viruses, integrating nucleicacids and other vectors or nucleic acids used to maintain or manipulatea nucleic acid insert of interest. Typically, the enriched nucleic acidsrepresent 15% or more of the number of nucleic acid inserts in thepopulation of recombinant backbone molecules. More typically, theenriched nucleic acids represent 50% or more of the number of nucleicacid inserts in the population of recombinant backbone molecules. In aone aspect, the enriched nucleic acids represent 90% or more of thenumber of nucleic acid inserts in the population of recombinant backbonemolecules.

“Plasmids” are designated by a lower case “p” preceded and/or followedby capital letters and/or numbers. The starting plasmids herein areeither commercially available, publicly available on an unrestrictedbasis, or can be constructed from available plasmids in accord withpublished procedures. In addition, equivalent plasmids to thosedescribed herein are known in the art and will be apparent to theordinarily skilled artisan. “Plasmids” can be commercially available,publicly available on an unrestricted basis, or can be constructed fromavailable plasmids in accord with published procedures. Equivalentplasmids to those described herein are known in the art and will beapparent to the ordinarily skilled artisan.

“Digestion” of DNA refers to catalytic cleavage of the DNA with arestriction enzyme that acts only at certain sequences in the DNA. Thevarious restriction enzymes used herein are commercially available andtheir reaction conditions, cofactors and other requirements were used aswould be known to the ordinarily skilled artisan. For analyticalpurposes, typically 1 μg of plasmid or DNA fragment is used with about 2units of enzyme in about 20 μl of buffer solution. For the purpose ofisolating DNA fragments for plasmid construction, typically 5 to 50 μgof DNA are digested with 20 to 250 units of enzyme in a larger volume.Appropriate buffers and substrate amounts for particular restrictionenzymes are specified by the manufacturer. Incubation times of about 1hour at 37° C. are ordinarily used, but may vary in accordance with thesupplier's instructions. After digestion, gel electrophoresis may beperformed to isolate the desired fragment.

“Hybridization” refers to the process by which a nucleic acid strandjoins with a complementary strand through base pairing. Hybridizationreactions can be sensitive and selective so that a particular sequenceof interest can be identified even in samples in which it is present atlow concentrations. Suitably stringent conditions can be defined by, forexample, the concentrations of salt or formamide in the prehybridizationand hybridization solutions, or by the hybridization temperature and arewell known in the art. In particular, stringency can be increased byreducing the concentration of salt, increasing the concentration offormamide, or raising the hybridization temperature. In alternativeaspects, nucleic acids of the invention are defined by their ability tohybridize under various stringency conditions (e.g., high, medium, andlow), as set forth herein.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5×SSPE, 0.3% SDSand 200 ug/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

Transcriptional and Translational Control Sequences

The invention provides nucleic acid (e.g., DNA) sequences of theinvention operatively linked to expression (e.g., transcriptional ortranslational) control sequence(s), e.g., promoters or enhancers, todirect or modulate RNA synthesis/expression. The expression controlsequence can be in an expression vector. Exemplary bacterial promotersinclude lacI, lacZ, T3, T7, gpt, lambda PR, PL and trp. Exemplaryeukaryotic promoters include CMV immediate early, HSV thymidine kinase,early and late SV40, LTRs from retrovirus, and mouse metallothionein I.A promoter sequence is “operably linked to” a coding sequence when RNApolymerase which initiates transcription at the promoter will transcribethe coding sequence into mRNA.

Promoters suitable for expressing a polypeptide in bacteria include theE. coli lac or trp promoters, the lad promoter, the lacZ promoter, theT3 promoter, the T7 promoter, the gpt promoter, the lambda PR promoter,the lambda PL promoter, promoters from operons encoding glycolyticenzymes such as 3-phosphoglycerate kinase (PGK), and the acidphosphatase promoter. Eukaryotic promoters include the CMV immediateearly promoter, the HSV thymidine kinase promoter, heat shock promoters,the early and late SV40 promoter, LTRs from retroviruses, and the mousemetallothionein-I promoter. Other promoters known to control expressionof genes in prokaryotic or eukaryotic cells or their viruses may also beused. Promoters suitable for expressing the polypeptide or fragmentthereof in bacteria include the E. coli lac or trp promoters, the lacIpromoter, the lacZ promoter, the T3 promoter, the T7 promoter, the gptpromoter, the lambda P_(R) promoter, the lambda P_(L) promoter,promoters from operons encoding glycolytic enzymes such as3-phosphoglycerate kinase (PGK) and the acid phosphatase promoter.Fungal promoters include the ∀ factor promoter. Eukaryotic promotersinclude the CMV immediate early promoter, the HSV thymidine kinasepromoter, heat shock promoters, the early and late SV40 promoter, LTRsfrom retroviruses and the mouse metallothionein-I promoter. Otherpromoters known to control expression of genes in prokaryotic oreukaryotic cells or their viruses may also be used.

Tissue-Specific Plant Promoters

The invention provides expression cassettes that can be expressed in atissue-specific manner, e.g., that can express an isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseof the invention in a tissue-specific manner. The invention alsoprovides plants or seeds that express an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase of theinvention in a tissue-specific manner. The tissue-specificity can beseed specific, stem specific, leaf specific, root specific, fruitspecific and the like.

In one aspect, a constitutive promoter such as the CaMV 35S promoter canbe used for expression in specific parts of the plant or seed orthroughout the plant. For example, for overexpression, a plant promoterfragment can be employed which will direct expression of a nucleic acidin some or all tissues of a plant, e.g., a regenerated plant. Suchpromoters are referred to herein as “constitutive” promoters and areactive under most environmental conditions and states of development orcell differentiation. Examples of constitutive promoters include thecauliflower mosaic virus (CaMV) 35S transcription initiation region, the1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, andother transcription initiation regions from various plant genes known tothose of skill. Such genes include, e.g., ACT11 from Arabidopsis (Huang(1996) Plant Mol. Biol. 33:125-139); Cat3 from Arabidopsis (GenBank No.U43147, Zhong (1996) Mol. Gen. Genet. 251:196-203); the gene encodingstearoyl-acyl carrier protein desaturase from Brassica napus (GenbankNo. X74782, Solocombe (1994) Plant Physiol. 104:1167-1176); GPc1 frommaize (GenBank No. X15596; Martinez (1989) J. Mol. Biol 208:551-565);the Gpc2 from maize (GenBank No. U45855, Manjunath (1997) Plant Mol.Biol. 33:97-112); plant promoters described in U.S. Pat. Nos. 4,962,028;5,633,440.

The invention uses tissue-specific or constitutive promoters derivedfrom viruses which can include, e.g., the tobamovirus subgenomicpromoter (Kumagai (1995) Proc. Natl. Acad. Sci. USA 92:1679-1683; therice tungro bacilliform virus (RTBV), which replicates only in phloemcells in infected rice plants, with its promoter which drives strongphloem-specific reporter gene expression; the cassava vein mosaic virus(CVMV) promoter, with highest activity in vascular elements, in leafmesophyll cells, and in root tips (Verdaguer (1996) Plant Mol. Biol.31:1129-1139).

Alternatively, the plant promoter may direct expression of anisomerase-, e.g., a racemase-, e.g., an amino acid racemase-, an alanineracemase-, and/or an epimerase-isomerase-, e.g., racemase-, e.g., aminoacid racemase-, alanine racemase-, and/or epimerase-expressing nucleicacid in a specific tissue, organ or cell type (i.e. tissue-specificpromoters) or may be otherwise under more precise environmental ordevelopmental control or under the control of an inducible promoter.Examples of environmental conditions that may affect transcriptioninclude anaerobic conditions, elevated temperature, the presence oflight, or sprayed with chemicals/hormones. For example, the inventionincorporates the drought-inducible promoter of maize (Busk (1997)supra); the cold, drought, and high salt inducible promoter from potato(Kirch (1997) Plant Mol. Biol. 33:897 909).

Tissue-specific promoters can promote transcription only within acertain time frame of developmental stage within that tissue. See, e.g.,Blazquez (1998) Plant Cell 10:791-800, characterizing the ArabidopsisLEAFY gene promoter. See also Cardon (1997) Plant J 12:367-77,describing the transcription factor SPL3, which recognizes a conservedsequence motif in the promoter region of the A. thaliana floral meristemidentity gene AP1; and Mandel (1995) Plant Molecular Biology, Vol. 29,pp 995-1004, describing the meristem promoter eIF4. Tissue specificpromoters which are active throughout the life cycle of a particulartissue can be used. In one aspect, the nucleic acids of the inventionare operably linked to a promoter active primarily only in cotton fibercells. In one aspect, the nucleic acids of the invention are operablylinked to a promoter active primarily during the stages of cotton fibercell elongation, e.g., as described by Rinehart (1996) supra. Thenucleic acids can be operably linked to the Fb12A gene promoter to bepreferentially expressed in cotton fiber cells (Ibid). See also, John(1997) Proc. Natl. Acad. Sci. USA 89:5769-5773; John, et al., U.S. Pat.Nos. 5,608,148 and 5,602,321, describing cotton fiber-specific promotersand methods for the construction of transgenic cotton plants.Root-specific promoters may also be used to express the nucleic acids ofthe invention. Examples of root-specific promoters include the promoterfrom the alcohol dehydrogenase gene (DeLisle (1990) Int. Rev. Cytol.123:39-60). Other promoters that can be used to express the nucleicacids of the invention include, e.g., ovule-specific, embryo-specific,endosperm-specific, integument-specific, seed coat-specific promoters,or some combination thereof; a leaf-specific promoter (see, e.g., Busk(1997) Plant J. 11:1285 1295, describing a leaf-specific promoter inmaize); the ORF13 promoter from Agrobacterium rhizogenes (which exhibitshigh activity in roots, see, e.g., Hansen (1997) supra); a maize pollenspecific promoter (see, e.g., Guerrero (1990) Mol. Gen. Genet. 224:161168); a tomato promoter active during fruit ripening, senescence andabscission of leaves and, to a lesser extent, of flowers can be used(see, e.g., Blume (1997) Plant J. 12:731 746); a pistil-specificpromoter from the potato SK2 gene (see, e.g., Ficker (1997) Plant Mol.Biol. 35:425 431); the Blec4 gene from pea, which is active in epidermaltissue of vegetative and floral shoot apices of transgenic alfalfamaking it a useful tool to target the expression of foreign genes to theepidermal layer of actively growing shoots or fibers; the ovule-specificBEL1 gene (see, e.g., Reiser (1995) Cell 83:735-742, GenBank No.U39944); and/or, the promoter in Klee, U.S. Pat. No. 5,589,583,describing a plant promoter region is capable of conferring high levelsof transcription in meristematic tissue and/or rapidly dividing cells.

Alternatively, plant promoters which are inducible upon exposure toplant hormones, such as auxins, are used to express the nucleic acids ofthe invention. For example, the invention can use the auxin-responseelements E1 promoter fragment (AuxREs) in the soybean (Glycine max L.)(Liu (1997) Plant Physiol. 115:397-407); the auxin-responsiveArabidopsis GST6 promoter (also responsive to salicylic acid andhydrogen peroxide) (Chen (1996) Plant J. 10: 955-966); theauxin-inducible parC promoter from tobacco (Sakai (1996) 37:906-913); aplant biotin response element (Streit (1997) Mol. Plant MicrobeInteract. 10:933-937); and, the promoter responsive to the stresshormone abscisic acid (Sheen (1996) Science 274:1900-1902).

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents whichcan be applied to the plant, such as herbicides or antibiotics. Forexample, the maize In2-2 promoter, activated by benzenesulfonamideherbicide safeners, can be used (De Veylder (1997) Plant Cell Physiol.38:568-577); application of different herbicide safeners inducesdistinct gene expression patterns, including expression in the root,hydathodes, and the shoot apical meristem. Coding sequence can be underthe control of, e.g., a tetracycline-inducible promoter, e.g., asdescribed with transgenic tobacco plants containing the Avena sativa L.(oat) arginine decarboxylase gene (Masgrau (1997) Plant J. 11:465-473);or, a salicylic acid-responsive element (Stange (1997) Plant J.11:1315-1324). Using chemically- (e.g., hormone- or pesticide-) inducedpromoters, i.e., promoter responsive to a chemical which can be appliedto the transgenic plant in the field, expression of a polypeptide of theinvention can be induced at a particular stage of development of theplant. Thus, the invention also provides for transgenic plantscontaining an inducible gene encoding for polypeptides of the inventionwhose host range is limited to target plant species, such as corn, rice,barley, wheat, potato or other crops, inducible at any stage ofdevelopment of the crop.

One of skill will recognize that a tissue-specific plant promoter maydrive expression of operably linked sequences in tissues other than thetarget tissue. Thus, a tissue-specific promoter is one that drivesexpression preferentially in the target tissue or cell type, but mayalso lead to some expression in other tissues as well.

The nucleic acids of the invention can also be operably linked to plantpromoters which are inducible upon exposure to chemicals reagents. Thesereagents include, e.g., herbicides, synthetic auxins, or antibioticswhich can be applied, e.g., sprayed, onto transgenic plants. Inducibleexpression of the an isomerase-, e.g., a racemase-, e.g., an amino acidracemase-, an alanine racemase-, and/or an epimerase-isomerase-, e.g.,racemase-, e.g., amino acid racemase-, alanine racemase-, and/orepimerase-producing nucleic acids of the invention will allow the growerto select plants with the optimal isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase expression and/oractivity. The development of plant parts can thus controlled. In thisway the invention provides the means to facilitate the harvesting ofplants and plant parts. For example, in various embodiments, the maizeIn2-2 promoter, activated by benzenesulfonamide herbicide safeners, isused (De Veylder (1997) Plant Cell Physiol. 38:568-577); application ofdifferent herbicide safeners induces distinct gene expression patterns,including expression in the root, hydathodes, and the shoot apicalmeristem. Coding sequences of the invention are also under the controlof a tetracycline-inducible promoter, e.g., as described with transgenictobacco plants containing the Avena sativa L. (oat) argininedecarboxylase gene (Masgrau (1997) Plant J. 11:465-473); or, a salicylicacid-responsive element (Stange (1997) Plant J. 11:1315-1324).

In some aspects, proper polypeptide expression may requirepolyadenylation region at the 3′-end of the coding region. Thepolyadenylation region can be derived from the natural gene, from avariety of other plant (or animal or other) genes, or from genes in theAgrobacterial T-DNA.

The term “plant” (e.g., as in a transgenic plant or plant seed of thisinvention, or plant promoter used in a vector of the invention) includeswhole plants, plant parts (e.g., leaves, stems, flowers, roots, etc.),plant protoplasts, seeds and plant cells and progeny of same; theclasses of plants that can be used to practice this invention (includingcompositions and methods) can be as broad as the class of higher plants,including plants amenable to transformation techniques, includingangiosperms (monocotyledonous and dicotyledonous plants), as well asgymnosperms; also including plants of a variety of ploidy levels,including polyploid, diploid, haploid and hemizygous states. As usedherein, the term “transgenic plant” includes plants or plant cells intowhich a heterologous nucleic acid sequence has been inserted, e.g., thenucleic acids and various recombinant constructs (e.g., expressioncassettes, such a vectors) of the invention. Transgenic plants of theinvention are also discussed, below.

Expression Vectors and Cloning Vehicles

The invention provides expression vectors and cloning vehiclescomprising nucleic acids of the invention, e.g., sequences encoding theisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention. Expression vectors andcloning vehicles of the invention can comprise viral particles,baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterialartificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul poxvirus, pseudorabies and derivatives of SV40), P1-based artificialchromosomes, yeast plasmids, yeast artificial chromosomes, and any othervectors specific for specific hosts of interest (such as bacillus,Aspergillus and yeast). Vectors of the invention can includechromosomal, non-chromosomal and synthetic DNA sequences. Large numbersof suitable vectors are known to those of skill in the art, and arecommercially available. Exemplary vectors are include: bacterial: pQEvectors (Qiagen), pBluescript plasmids, pNH vectors, (lambda-ZAP vectors(Stratagene); ptrc99a, pKK223-3, pDR540, pRIT2T (Pharmacia); Eukaryotic:pXT1, pSG5 (Stratagene), pSVK3, pBPV, pMSG, pSVLSV40 (Pharmacia).However, any other plasmid or other vector may be used so long as theyare replicable and viable in the host. Low copy number or high copynumber vectors may be employed with the present invention.

The expression vector can comprise a promoter, a ribosome binding sitefor translation initiation and a transcription terminator. The vectormay also include appropriate sequences for amplifying expression.Mammalian expression vectors can comprise an origin of replication, anynecessary ribosome binding sites, a polyadenylation site, splice donorand acceptor sites, transcriptional termination sequences, and 5′flanking non-transcribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required non-transcribed genetic elements.

In one aspect, the expression vectors contain one or more selectablemarker genes to permit selection of host cells containing the vector.Such selectable markers include genes encoding dihydrofolate reductaseor genes conferring neomycin resistance for eukaryotic cell culture,genes conferring tetracycline or ampicillin resistance in E. coli, andthe S. cerevisiae TRP1 gene. Promoter regions can be selected from anydesired gene using chloramphenicol transferase (CAT) vectors or othervectors with selectable markers.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells can also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, and theadenovirus enhancers.

A nucleic acid sequence can be inserted into a vector by a variety ofprocedures. In general, the sequence is ligated to the desired positionin the vector following digestion of the insert and the vector withappropriate restriction endonucleases. Alternatively, blunt ends in boththe insert and the vector may be ligated. A variety of cloningtechniques are known in the art, e.g., as described in Ausubel andSambrook. Such procedures and others are deemed to be within the scopeof those skilled in the art.

The vector can be in the form of a plasmid, a viral particle, or aphage. Other vectors include chromosomal, non-chromosomal and syntheticDNA sequences, derivatives of SV40; bacterial plasmids, phage DNA,baculovirus, yeast plasmids, vectors derived from combinations ofplasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl poxvirus, and pseudorabies. A variety of cloning and expression vectors foruse with prokaryotic and eukaryotic hosts are described by, e.g.,Sambrook.

Particular bacterial vectors which can be used include the commerciallyavailable plasmids comprising genetic elements of the well known cloningvector pBR322 (ATCC 37017), pKK223-3 (Pharmacia Fine Chemicals, Uppsala,Sweden), GEM1 (Promega Biotec, Madison, Wis., USA) pQE70, pQE60, pQE-9(Qiagen), pD10, psiX174 pBluescript II KS, pNH8A, pNH16a, pNH18A, pNH46A(Stratagene), ptrc99a, pKK223-3, pKK233-3, DR540, pRIT5 (Pharmacia),pKK232-8 and pCM7. Particular eukaryotic vectors include pSV2CAT, pOG44,pXT1, pSG (Stratagene) pSVK3, pBPV, pMSG, and pSVL (Pharmacia). However,any other vector may be used as long as it is replicable and viable inthe host cell.

The nucleic acids of the invention can be expressed in expressioncassettes, vectors or viruses and transiently or stably expressed inplant cells and seeds. One exemplary transient expression system usesepisomal expression systems, e.g., cauliflower mosaic virus (CaMV) viralRNA generated in the nucleus by transcription of an episomalmini-chromosome containing supercoiled DNA, see, e.g., Covey (1990)Proc. Natl. Acad. Sci. USA 87:1633-1637. Alternatively, codingsequences, i.e., all or sub-fragments of sequences of the invention canbe inserted into a plant host cell genome becoming an integral part ofthe host chromosomal DNA. Sense or antisense transcripts can beexpressed in this manner. A vector comprising the sequences (e.g.,promoters or coding regions) from nucleic acids of the invention cancomprise a marker gene that confers a selectable phenotype on a plantcell or a seed. For example, the marker may encode biocide resistance,particularly antibiotic resistance, such as resistance to kanamycin,G418, bleomycin, hygromycin, or herbicide resistance, such as resistanceto chlorosulfuron or Basta.

Expression vectors capable of expressing nucleic acids and proteins inplants are well known in the art, and can include, e.g., vectors fromAgrobacterium spp., potato virus X (see, e.g., Angell (1997) EMBO J.16:3675-3684), tobacco mosaic virus (see, e.g., Casper (1996) Gene173:69-73), tomato bushy stunt virus (see, e.g., Hillman (1989) Virology169:42-50), tobacco etch virus (see, e.g., Dolja (1997) Virology234:243-252), bean golden mosaic virus (see, e.g., Morinaga (1993)Microbiol Immunol. 37:471-476), cauliflower mosaic virus (see, e.g.,Cecchini (1997) Mol. Plant Microbe Interact. 10:1094-1101), maize Ac/Dstransposable element (see, e.g., Rubin (1997) Mol. Cell. Biol.17:6294-6302; Kunze (1996) Curr. Top. Microbiol. Immunol. 204:161-194),and the maize suppressor-mutator (Spm) transposable element (see, e.g.,Schlappi (1996) Plant Mol. Biol. 32:717-725); and derivatives thereof.

In one aspect, the expression vector can have two replication systems toallow it to be maintained in two organisms, for example in mammalian orinsect cells for expression and in a prokaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector can contain at least one sequence homologous to thehost cell genome. It can contain two homologous sequences which flankthe expression construct. The integrating vector can be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

Expression vectors of the invention may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed, e.g., genes which render the bacteria resistant to drugssuch as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycinand tetracycline. Selectable markers can also include biosyntheticgenes, such as those in the histidine, tryptophan and leucinebiosynthetic pathways.

The DNA sequence in the expression vector is operatively linked to anappropriate expression control sequence(s) (promoter) to direct RNAsynthesis. Particular named bacterial promoters include lacI, lacZ, T3,T7, gpt, lambda P_(R), P_(L) and trp. Eukaryotic promoters include CMVimmediate early, HSV thymidine kinase, early and late SV40, LTRs fromretrovirus and mouse metallothionein-I. Selection of the appropriatevector and promoter is well within the level of ordinary skill in theart. The expression vector also contains a ribosome binding site fortranslation initiation and a transcription terminator. The vector mayalso include appropriate sequences for amplifying expression. Promoterregions can be selected from any desired gene using chloramphenicoltransferase (CAT) vectors or other vectors with selectable markers. Inaddition, the expression vectors preferably contain one or moreselectable marker genes to provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture, or such as tetracycline orampicillin resistance in E. coli.

Mammalian expression vectors may also comprise an origin of replication,any necessary ribosome binding sites, a polyadenylation site, splicedonor and acceptor sites, transcriptional termination sequences and 5′flanking nontranscribed sequences. In some aspects, DNA sequencesderived from the SV40 splice and polyadenylation sites may be used toprovide the required nontranscribed genetic elements.

Vectors for expressing the polypeptide or fragment thereof in eukaryoticcells may also contain enhancers to increase expression levels.Enhancers are cis-acting elements of DNA, usually from about 10 to about300 bp in length that act on a promoter to increase its transcription.Examples include the SV40 enhancer on the late side of the replicationorigin by 100 to 270, the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin and theadenovirus enhancers.

In addition, the expression vectors typically contain one or moreselectable marker genes to permit selection of host cells containing thevector. Such selectable markers include genes encoding dihydrofolatereductase or genes conferring neomycin resistance for eukaryotic cellculture, genes conferring tetracycline or ampicillin resistance in E.coli and the S. cerevisiae TRP1 gene.

In some aspects, the nucleic acid encoding one of the polypeptides ofthe invention and sequences substantially identical thereto, orfragments comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50,75, 100, or 150 consecutive amino acids thereof is assembled inappropriate phase with a leader sequence capable of directing secretionof the translated polypeptide or fragment thereof. The nucleic acid canencode a fusion polypeptide in which one of the polypeptides of theinvention and sequences substantially identical thereto, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof is fused to heterologous peptides orpolypeptides, such as N-terminal identification peptides which impartdesired characteristics, such as increased stability or simplifiedpurification.

The appropriate DNA sequence may be inserted into the vector by avariety of procedures. In general, the DNA sequence is ligated to thedesired position in the vector following digestion of the insert and thevector with appropriate restriction endonucleases. Alternatively, bluntends in both the insert and the vector may be ligated. A variety ofcloning techniques are disclosed in Ausubel et al. Current Protocols inMolecular Biology, John Wiley 503 Sons, Inc. 1997 and Sambrook et al.,Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring HarborLaboratory Press (1989. Such procedures and others are deemed to bewithin the scope of those skilled in the art.

The vector may be, for example, in the form of a plasmid, a viralparticle, or a phage. Other vectors include chromosomal, nonchromosomaland synthetic DNA sequences, derivatives of SV40; bacterial plasmids,phage DNA, baculovirus, yeast plasmids, vectors derived fromcombinations of plasmids and phage DNA, viral DNA such as vaccinia,adenovirus, fowl pox virus and pseudorabies. A variety of cloning andexpression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor, N.Y., (1989).

Host Cells and Transformed Cells

The invention also provides transformed cells comprising a nucleic acidsequence of the invention, e.g., a sequence encoding an isomerase, e.g.,a racemase, e.g., an amino acid racemase, an alanine racemase, and/or anepimerase of the invention, or a vector of the invention. The host cellmay be any of the host cells familiar to those skilled in the art,including prokaryotic cells, eukaryotic cells, such as bacterial cells,fungal cells, yeast cells, mammalian cells, insect cells, or plantcells. Exemplary bacterial cells include any species within the generaEscherichia, Bacillus, Streptomyces, Salmonella, Pseudomonas andStaphylococcus, including, e.g., Escherichia coli, Lactococcus lactis,Bacillus subtilis, Bacillus cereus, Salmonella typhimurium, Pseudomonasfluorescens. Exemplary fungal cells include any species of Aspergillus.Exemplary yeast cells include any species of Pichia, Saccharomyces,Schizosaccharomyces, or Schwanniomyces, including Pichia pastoris,Saccharomyces cerevisiae, or Schizosaccharomyces pombe. Exemplary insectcells include any species of Spodoptera or Drosophila, includingDrosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO,COS or Bowes melanoma or any mouse or human cell line. The selection ofan appropriate host is within the abilities of those skilled in the art.Techniques for transforming a wide variety of higher plant species arewell known and described in the technical and scientific literature.See, e.g., Weising (1988) Ann. Rev. Genet. 22:421-477; U.S. Pat. No.5,750,870.

The vector can be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

In one aspect, the nucleic acids or vectors of the invention areintroduced into the cells for screening, thus, the nucleic acids enterthe cells in a manner suitable for subsequent expression of the nucleicacid. The method of introduction is largely dictated by the targetedcell type. Exemplary methods include CaPO₄ precipitation, liposomefusion, lipofection (e.g., LIPOFECTIN™), electroporation, viralinfection, etc. The candidate nucleic acids may stably integrate intothe genome of the host cell (for example, with retroviral introduction)or may exist either transiently or stably in the cytoplasm (i.e. throughthe use of traditional plasmids, utilizing standard regulatorysequences, selection markers, etc.). As many pharmaceutically importantscreens require human or model mammalian cell targets, retroviralvectors capable of transfecting such targets are can be used.

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells can be harvested by centrifugation, disrupted by physical orchemical means, and the resulting crude extract is retained for furtherpurification. Microbial cells employed for expression of proteins can bedisrupted by any convenient method, including freeze-thaw cycling,sonication, mechanical disruption, or use of cell lysing agents. Suchmethods are well known to those skilled in the art. The expressedpolypeptide or fragment thereof can be recovered and purified fromrecombinant cell cultures by methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Protein refolding steps can be used, asnecessary, in completing configuration of the polypeptide. If desired,high performance liquid chromatography (HPLC) can be employed for finalpurification steps.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Cell-free translation systems can also be employed to produce apolypeptide of the invention. Cell-free translation systems can usemRNAs transcribed from a DNA construct comprising a promoter operablylinked to a nucleic acid encoding the polypeptide or fragment thereof.In some aspects, the DNA construct may be linearized prior to conductingan in vitro transcription reaction. The transcribed mRNA is thenincubated with an appropriate cell-free translation extract, such as arabbit reticulocyte extract, to produce the desired polypeptide orfragment thereof.

The expression vectors can contain one or more selectable marker genesto provide a phenotypic trait for selection of transformed host cellssuch as dihydrofolate reductase or neomycin resistance for eukaryoticcell culture, or such as tetracycline or ampicillin resistance in E.coli.

Host cells containing the polynucleotides of interest, e.g., nucleicacids of the invention, can be cultured in conventional nutrient mediamodified as appropriate for activating promoters, selectingtransformants or amplifying genes. The culture conditions, such astemperature, pH and the like, are those previously used with the hostcell selected for expression and will be apparent to the ordinarilyskilled artisan. The clones which are identified as having the specifiedenzyme activity may then be sequenced to identify the polynucleotidesequence encoding an enzyme having the enhanced activity.

The invention provides a method for overexpressing a recombinantisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase in a cell comprising expressing a vector comprising anucleic acid of the invention, e.g., a nucleic acid comprising a nucleicacid sequence with at least about 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more sequence identity to a sequence of the invention over aregion of at least about 100 residues, wherein the sequence identitiesare determined by analysis with a sequence comparison algorithm or byvisual inspection, or, a nucleic acid that hybridizes under stringentconditions to a nucleic acid sequence of the invention, or a subsequencethereof. The overexpression can be effected by any means, e.g., use of ahigh activity promoter, a dicistronic vector or by gene amplification ofthe vector.

The nucleic acids of the invention can be expressed, or overexpressed,in any in vitro or in vivo expression system. Any cell culture systemscan be employed to express, or over-express, recombinant protein,including bacterial, insect, yeast, fungal or mammalian cultures.Over-expression can be effected by appropriate choice of promoters,enhancers, vectors (e.g., use of replicon vectors, dicistronic vectors(see, e.g., Gurtu (1996) Biochem. Biophys. Res. Commun 229:295-8),media, culture systems and the like. In one aspect, gene amplificationusing selection markers, e.g., glutamine synthetase (see, e.g., Sanders(1987) Dev. Biol. Stand. 66:55-63), in cell systems are used tooverexpress the polypeptides of the invention.

Additional details regarding this approach are in the public literatureand/or are known to the skilled artisan. In a particular non-limitingexemplification, such publicly available literature includes, forexample: Appl Environ Microbiol. 2004 June; 70(6):3298-304; BiotechnolBioeng. 2007 Nov. 1; 98(4):812-24 and FEMS Microbiol Lett. 2001 Mar. 15;196(2):93-8, although these references do not teach the inventiveenzymes of the instant application.

The host cell may be any of the host cells familiar to those skilled inthe art, including prokaryotic cells, eukaryotic cells, mammalian cells,insect cells, or plant cells. As representative examples of appropriatehosts, there may be mentioned: bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, fungal cells, such as Aspergillus, yeast such as anyspecies of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces,including Pichia pastoris, Saccharomyces cerevisiae, orSchizosaccharomyces pombe, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art.

The vector may be introduced into the host cells using any of a varietyof techniques, including transformation, transfection, transduction,viral infection, gene guns, or Ti-mediated gene transfer. Particularmethods include calcium phosphate transfection, DEAE-Dextran mediatedtransfection, lipofection, or electroporation (Davis, L., Dibner, M.,Battey, I., Basic Methods in Molecular Biology, (1986)).

Where appropriate, the engineered host cells can be cultured inconventional nutrient media modified as appropriate for activatingpromoters, selecting transformants or amplifying the genes of theinvention. Following transformation of a suitable host strain and growthof the host strain to an appropriate cell density, the selected promotermay be induced by appropriate means (e.g., temperature shift or chemicalinduction) and the cells may be cultured for an additional period toallow them to produce the desired polypeptide or fragment thereof.

Cells are typically harvested by centrifugation, disrupted by physicalor chemical means and the resulting crude extract is retained forfurther purification. Microbial cells employed for expression ofproteins can be disrupted by any convenient method, includingfreeze-thaw cycling, sonication, mechanical disruption, or use of celllysing agents. Such methods are well known to those skilled in the art.The expressed polypeptide or fragment thereof can be recovered andpurified from recombinant cell cultures by methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Protein refolding steps can beused, as necessary, in completing configuration of the polypeptide. Ifdesired, high performance liquid chromatography (HPLC) can be employedfor final purification steps.

Various mammalian cell culture systems can also be employed to expressrecombinant protein. Examples of mammalian expression systems includethe COS-7 lines of monkey kidney fibroblasts (described by Gluzman,Cell, 23:175, 1981) and other cell lines capable of expressing proteinsfrom a compatible vector, such as the C127, 3T3, CHO, HeLa and BHK celllines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. Dependingupon the host employed in a recombinant production procedure, thepolypeptides produced by host cells containing the vector may beglycosylated or may be non-glycosylated. Polypeptides of the inventionmay or may not also include an initial methionine amino acid residue.

Alternatively, the polypeptides of amino acid sequences of theinvention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 consecutive amino acids thereof can besynthetically produced by conventional peptide synthesizers. In otheraspects, fragments or portions of the polypeptides may be employed forproducing the corresponding full-length polypeptide by peptidesynthesis; therefore, the fragments may be employed as intermediates forproducing the full-length polypeptides.

Cell-free translation systems can also be employed to produce one of thepolypeptides of amino acid sequences of the invention, or fragmentscomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof using mRNAs transcribed from a DNAconstruct comprising a promoter operably linked to a nucleic acidencoding the polypeptide or fragment thereof. In some aspects, the DNAconstruct may be linearized prior to conducting an in vitrotranscription reaction. The transcribed mRNA is then incubated with anappropriate cell-free translation extract, such as a rabbit reticulocyteextract, to produce the desired polypeptide or fragment thereof.

Amplification of Nucleic Acids

In practicing the invention, nucleic acids of the invention and nucleicacids encoding the isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention, ormodified nucleic acids of the invention, can be reproduced byamplification. Amplification can also be used to clone or modify thenucleic acids of the invention. Thus, the invention providesamplification primer sequence pairs for amplifying nucleic acids of theinvention. One of skill in the art can design amplification primersequence pairs for any part of or the full length of these sequences.

In one aspect, the invention provides a nucleic acid amplified by aprimer pair of the invention, e.g., a primer pair as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and about the first(the 5′) 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand.

The invention provides an amplification primer sequence pair foramplifying a nucleic acid encoding a polypeptide having an isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase activity, wherein the primer pair is capable of amplifying anucleic acid comprising a sequence of the invention, or fragments orsubsequences thereof. One or each member of the amplification primersequence pair can comprise an oligonucleotide comprising at least about10 to 50 consecutive bases of the sequence, or about 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, or 25 consecutive bases of the sequence.The invention provides amplification primer pairs, wherein the primerpair comprises a first member having a sequence as set forth by aboutthe first (the 5′) 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,or 25 residues of a nucleic acid of the invention, and a second memberhaving a sequence as set forth by about the first (the 5′) 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 residues of thecomplementary strand of the first member. The invention providesisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases generated by amplification, e.g.,polymerase chain reaction (PCR), using an amplification primer pair ofthe invention. The invention provides methods of making an isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase by amplification, e.g., polymerase chain reaction (PCR), usingan amplification primer pair of the invention. In one aspect, theamplification primer pair amplifies a nucleic acid from a library, e.g.,a gene library, such as an environmental library.

Amplification reactions can also be used to quantify the amount ofnucleic acid in a sample (such as the amount of message in a cellsample), label the nucleic acid (e.g., to apply it to an array or ablot), detect the nucleic acid, or quantify the amount of a specificnucleic acid in a sample. In one aspect of the invention, messageisolated from a cell or a cDNA library are amplified.

The skilled artisan can select and design suitable oligonucleotideamplification primers. Amplification methods are also well known in theart, and include, e.g., polymerase chain reaction, PCR (see, e.g., PCRPROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, AcademicPress, N.Y. (1990) and PCR STRATEGIES (1995), ed. Innis, Academic Press,Inc., N.Y., ligase chain reaction (LCR) (see, e.g., Wu (1989) Genomics4:560; Landegren (1988) Science 241:1077; Barringer (1990) Gene 89:117);transcription amplification (see, e.g., Kwoh (1989) Proc. Natl. Acad.Sci. USA 86:1173); and, self-sustained sequence replication (see, e.g.,Guatelli (1990) Proc. Natl. Acad. Sci. USA 87:1874); Q Beta replicaseamplification (see, e.g., Smith (1997) J. Clin. Microbiol.35:1477-1491), automated Q-beta replicase amplification assay (see,e.g., Burg (1996) Mol. Cell. Probes 10:257-271) and other RNA polymerasemediated techniques (e.g., NASBA, Cangene, Mississauga, Ontario); seealso Berger (1987) Methods Enzymol. 152:307-316; Sambrook; Ausubel; U.S.Pat. Nos. 4,683,195 and 4,683,202; Sooknanan (1995) Biotechnology13:563-564.

Determining the Degree of Sequence Identity

The invention provides nucleic acids comprising sequences having atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary nucleic acid of the invention(as defined above) over a region of at least about 50, 75, 100, 150,200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850,900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450,1500, 1550 or more, residues. The invention provides polypeptidescomprising sequences having at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, 99%, or more, or complete (100%) sequence identity to an exemplarypolypeptide of the invention. The extent of sequence identity (homology)may be determined using any computer program and associated parameters,including those described herein, such as BLAST 2.2.2. or FASTA version3.0t78, with the default parameters.

As used herein, the terms “computer,” “computer program” and “processor”are used in their broadest general contexts and incorporate all suchdevices, as described in detail, below. A “coding sequence of” or a“sequence encodes” a particular polypeptide or protein, is a nucleicacid sequence which is transcribed and translated into a polypeptide orprotein when placed under the control of appropriate regulatorysequences.

The phrase “substantially identical” in the context of two nucleic acidsor polypeptides, refers to two or more sequences that have, e.g., atleast about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more nucleotide oramino acid residue (sequence) identity, when compared and aligned formaximum correspondence, as measured using one of the known sequencecomparison algorithms or by visual inspection. Typically, thesubstantial identity exists over a region of at least about 100 residuesand most commonly the sequences are substantially identical over atleast about 150-200 residues. In some aspects, the sequences aresubstantially identical over the entire length of the coding regions.

Additionally a “substantially identical” amino acid sequence is asequence that differs from a reference sequence by one or moreconservative or non-conservative amino acid substitutions, deletions, orinsertions, particularly when such a substitution occurs at a site thatis not the active site of the molecule and provided that the polypeptideessentially retains its functional properties. A conservative amino acidsubstitution, for example, substitutes one amino acid for another of thesame class (e.g., substitution of one hydrophobic amino acid, such asisoleucine, valine, leucine, or methionine, for another, or substitutionof one polar amino acid for another, such as substitution of argininefor lysine, glutamic acid for aspartic acid or glutamine forasparagine). One or more amino acids can be deleted, for example, froman isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase polypeptide, resulting in modification of thestructure of the polypeptide, without significantly altering itsbiological activity. For example, amino- or carboxyl-terminal aminoacids that are not required for isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase biological activitycan be removed. Modified polypeptide sequences of the invention can beassayed for isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase biological activity by any number ofmethods, including contacting the modified polypeptide sequence with anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase substrate and determining whether the modifiedpolypeptide decreases the amount of specific substrate in the assay orincreases the bioproducts of the enzymatic reaction of a functionalisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase polypeptide with the substrate.

Nucleic acid sequences of the invention can comprise at least 10, 15,20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 consecutivenucleotides of an exemplary sequence of the invention and sequencessubstantially identical thereto. Nucleic acid sequences of the inventioncan comprise homologous sequences and fragments of nucleic acidsequences and sequences substantially identical thereto, refer to asequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or moresequence identity (homology) to these sequences. Homology may bedetermined using any of the computer programs and parameters describedherein, including FASTA version 3.0t78 with the default parameters.Homologous sequences also include RNA sequences in which uridinesreplace the thymines in the nucleic acid sequences of the invention. Thehomologous sequences may be obtained using any of the proceduresdescribed herein or may result from the correction of a sequencingerror. It will be appreciated that the nucleic acid sequences of theinvention and sequences substantially identical thereto, can berepresented in the traditional single character format (See the insideback cover of Stryer, Lubert. Biochemistry, 3rd Ed., W. H Freeman & Co.,New York.) or in any other format which records the identity of thenucleotides in a sequence.

Various sequence comparison programs identified elsewhere in this patentspecification are particularly contemplated for use in this aspect ofthe invention. Protein and/or nucleic acid sequence homologies may beevaluated using any of the variety of sequence comparison algorithms andprograms known in the art. Such algorithms and programs include, but areby no means limited to, TBLASTN, BLASTP, FASTA, TFASTA and CLUSTALW(Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988;Altschul et al., J. Mol. Biol. 215(3):403-410, 1990; Thompson et al.,Nucleic Acids Res. 22(2):4673-4680, 1994; Higgins et al., MethodsEnzymol. 266:383-402, 1996; Altschul et al., J. Mol. Biol.215(3):403-410, 1990; Altschul et al., Nature Genetics 3:266-272, 1993).

Homology or identity is often measured using sequence analysis software(e.g., Sequence Analysis Software Package of the Genetics ComputerGroup, University of Wisconsin Biotechnology Center, 1710 UniversityAvenue, Madison, Wis. 53705). Such software matches similar sequences byassigning degrees of homology to various deletions, substitutions andother modifications. The terms “homology” and “identity” in the contextof two or more nucleic acids or polypeptide sequences, refer to two ormore sequences or subsequences that are the same or have a specifiedpercentage of amino acid residues or nucleotides that are the same whencompared and aligned for maximum correspondence over a comparison windowor designated region as measured using any number of sequence comparisonalgorithms or by manual alignment and visual inspection.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencefor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol 48:443, 1970, bythe search for similarity method of person & Lipman, Proc. Nat'l. Acad.Sci. USA 85:2444, 1988, by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin GeneticsSoftware Package, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by manual alignment and visual inspection. Other algorithmsfor determining homology or identity include, for example, in additionto a BLAST program (Basic Local Alignment Search Tool at the NationalCenter for Biological Information), ALIGN, AMAS (Analysis of MultiplyAligned Sequences), AMPS (Protein Multiple Sequence Alignment), ASSET(Aligned Segment Statistical Evaluation Tool), BANDS, BESTSCOR, BIOSCAN(Biological Sequence Comparative Analysis Node), BLIMPS (BLocks IMProvedSearcher), FASTA, Intervals & Points, BMB, CLUSTAL V, CLUSTAL W,CONSENSUS, LCONSENSUS, WCONSENSUS, Smith-Waterman algorithm, DARWIN, LasVegas algorithm, FNAT (Forced Nucleotide Alignment Tool), Framealign,Framesearch, DYNAMIC, FILTER, FSAP (Fristensky Sequence AnalysisPackage), GAP (Global Alignment Program), GENAL, GIBBS, GenQuest, ISSC(Sensitive Sequence Comparison), LALIGN (Local Sequence Alignment), LCP(Local Content Program), MACAW (Multiple Alignment Construction &Analysis Workbench), MAP (Multiple Alignment Program), MBLKP, MBLKN,PIMA (Pattern-Induced Multi-sequence Alignment), SAGA (SequenceAlignment by Genetic Algorithm) and WHAT-IF. Such alignment programs canalso be used to screen genome databases to identify polynucleotidesequences having substantially identical sequences. A number of genomedatabases are available, for example, a substantial portion of the humangenome is available as part of the Human Genome Sequencing Project. Atleast twenty-one other genomes have already been sequenced, including,for example, M. genitalium (Fraser et al., 1995), M. jannaschii (Butt etal., 1996), H. influenzae (Fleischmann et al., 1995), E. coli (Blattneret al., 1997) and yeast (S. cerevisiae) (Mewes et al., 1997) and D.melanogaster (Adams et al., 2000). Significant progress has also beenmade in sequencing the genomes of model organism, such as mouse, C.elegans and Arabadopsis sp. Several databases containing genomicinformation annotated with some functional information are maintained bydifferent organization and are accessible via the internet

One example of a useful algorithm is BLAST and BLAST 2.0 algorithms,which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402,1977 and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold (Altschul et al., supra). Theseinitial neighborhood word hits act as seeds for initiating searches tofind longer HSPs containing them. The word hits are extended in bothdirections along each sequence for as far as the cumulative alignmentscore can be increased. Cumulative scores are calculated using, fornucleotide sequences, the parameters M (reward score for a pair ofmatching residues; always >0). For amino acid sequences, a scoringmatrix is used to calculate the cumulative score. Extension of the wordhits in each direction are halted when: the cumulative alignment scorefalls off by the quantity X from its maximum achieved value; thecumulative score goes to zero or below, due to the accumulation of oneor more negative-scoring residue alignments; or the end of eithersequence is reached. The BLAST algorithm parameters W, T and X determinethe sensitivity and speed of the alignment. The BLASTN program (fornucleotide sequences) uses as defaults a wordlength (W) of 11, anexpectation (E) of 10, M=5, N=−4 and a comparison of both strands. Foramino acid sequences, the BLASTP program uses as defaults a wordlengthof 3 and expectations (E) of 10 and the BLOSUM62 scoring matrix (seeHenikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915, 1989)alignments (B) of 50, expectation (E) of 10, M=5, N=−4 and a comparisonof both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Natl. Acad. Sci. USA 90:5873, 1993). One measure of similarity providedby BLAST algorithm is the smallest sum probability (P(N)), whichprovides an indication of the probability by which a match between twonucleotide or amino acid sequences would occur by chance. For example, anucleic acid is considered similar to a references sequence if thesmallest sum probability in a comparison of the test nucleic acid to thereference nucleic acid is less than about 0.2, more preferably less thanabout 0.01 and most preferably less than about 0.001.

In one aspect, protein and nucleic acid sequence homologies areevaluated using the Basic Local Alignment Search Tool (“BLAST”) Inparticular, five specific BLAST programs are used to perform thefollowing task:

-   -   (1) BLASTP and BLAST3 compare an amino acid query sequence        against a protein sequence database;    -   (2) BLASTN compares a nucleotide query sequence against a        nucleotide sequence database;    -   (3) BLASTX compares the six-frame conceptual translation        products of a query nucleotide sequence (both strands) against a        protein sequence database;    -   (4) TBLASTN compares a query protein sequence against a        nucleotide sequence database translated in all six reading        frames (both strands); and    -   (5) TBLASTX compares the six-frame translations of a nucleotide        query sequence against the six-frame translations of a        nucleotide sequence database.

The BLAST programs identify homologous sequences by identifying similarsegments, which are referred to herein as “high-scoring segment pairs,”between a query amino or nucleic acid sequence and a test sequence whichis preferably obtained from a protein or nucleic acid sequence database.High-scoring segment pairs are preferably identified (i.e., aligned) bymeans of a scoring matrix, many of which are known in the art.Preferably, the scoring matrix used is the BLOSUM62 matrix (Gonnet etal., Science 256:1443-1445, 1992; Henikoff and Henikoff, Proteins17:49-61, 1993). Less preferably, the PAM or PAM250 matrices may also beused (see, e.g., Schwartz and Dayhoff, eds., 1978, Matrices forDetecting Distance Relationships: Atlas of Protein Sequence andStructure, Washington: National Biomedical Research Foundation). BLASTprograms are accessible through the U.S. National Library of Medicine.

The parameters used with the above algorithms may be adapted dependingon the sequence length and degree of homology studied. In some aspects,the parameters may be the default parameters used by the algorithms inthe absence of instructions from the user.

Computer Systems and Computer Program Products

To determine and identify sequence identities, structural homologies,motifs and the like in silico, a nucleic acid or polypeptide sequence ofthe invention can be stored, recorded, and manipulated on any mediumwhich can be read and accessed by a computer.

Accordingly, the invention provides computers, computer systems,computer readable mediums, computer programs products and the likerecorded or stored thereon the nucleic acid and polypeptide sequences ofthe invention. As used herein, the words “recorded” and “stored” referto a process for storing information on a computer medium. A skilledartisan can readily adopt any known methods for recording information ona computer readable medium to generate manufactures comprising one ormore of the nucleic acid and/or polypeptide sequences of the invention.

The polypeptides of the invention include the exemplary sequences of theinvention, and sequences substantially identical thereto, and fragmentsof any of the preceding sequences. Substantially identical, orhomologous, polypeptide sequences refer to a polypeptide sequence havingat least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more, or complete(100%) sequence identity to an exemplary sequence of the invention,e.g., a polypeptide sequences of the invention.

Homology may be determined using any of the computer programs andparameters described herein, including FASTA version 3.0t78 with thedefault parameters or with any modified parameters. The homologoussequences may be obtained using any of the procedures described hereinor may result from the correction of a sequencing error. The polypeptidefragments comprise at least about 10, 15, 20, 25, 30, 35, 40, 45, 50,75, 100, 150, 200, 250, 300, 350, 400, 450, 500 or more consecutiveamino acids of the polypeptides of the invention and sequencessubstantially identical thereto. It will be appreciated that thepolypeptide codes of amino acid sequences of the invention and sequencessubstantially identical thereto, can be represented in the traditionalsingle character format or three letter format (See Stryer, Lubert.Biochemistry, 3rd Ed., supra) or in any other format which relates theidentity of the polypeptides in a sequence.

A nucleic acid or polypeptide sequence of the invention can be stored,recorded and manipulated on any medium which can be read and accessed bya computer. As used herein, the words “recorded” and “stored” refer to aprocess for storing information on a computer medium. A skilled artisancan readily adopt any of the presently known methods for recordinginformation on a computer readable medium to generate manufacturescomprising one or more of the nucleic acid sequences of the inventionand sequences substantially identical thereto, one or more of thepolypeptide sequences of the invention and sequences substantiallyidentical thereto. Another aspect of the invention is a computerreadable medium having recorded thereon at least 2, 5, 10, 15, or 20 ormore nucleic acid sequences of the invention and sequences substantiallyidentical thereto.

Another aspect of the invention is a computer readable medium havingrecorded thereon one or more of the nucleic acid sequences of theinvention and sequences substantially identical thereto. Another aspectof the invention is a computer readable medium having recorded thereonone or more of the polypeptide sequences of the invention and sequencessubstantially identical thereto. Another aspect of the invention is acomputer readable medium having recorded thereon at least 2, 5, 10, 15,or 20 or more of the sequences as set forth above.

Computer readable media include magnetically readable media, opticallyreadable media, electronically readable media and magnetic/opticalmedia. For example, the computer readable media may be a hard disk, afloppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD),Random Access Memory (RAM), or Read Only Memory (ROM) as well as othertypes of other media known to those skilled in the art.

Aspects of the invention include systems (e.g., internet based systems),particularly computer systems which store and manipulate the sequenceinformation described herein. One example of a computer system 100 isillustrated in block diagram form in FIG. 1. As used herein, “a computersystem” refers to the hardware components, software components and datastorage components used to analyze a nucleotide sequence of a nucleicacid sequence of the invention and sequences substantially identicalthereto, or a polypeptide sequence as set forth in the amino acidsequences of the invention. The computer system 100 typically includes aprocessor for processing, accessing and manipulating the sequence data.The processor 105 can be any well-known type of central processing unit,such as, for example, the Pentium III from Intel Corporation, or similarprocessor from Sun, Motorola, Compaq, AMD or International BusinessMachines.

Typically the computer system 100 is a general purpose system thatcomprises the processor 105 and one or more internal data storagecomponents 110 for storing data and one or more data retrieving devicesfor retrieving the data stored on the data storage components. A skilledartisan can readily appreciate that any one of the currently availablecomputer systems are suitable.

In one particular aspect, the computer system 100 includes a processor105 connected to a bus which is connected to a main memory 115(preferably implemented as RAM) and one or more internal data storagedevices 110, such as a hard drive and/or other computer readable mediahaving data recorded thereon. In some aspects, the computer system 100further includes one or more data retrieving device 118 for reading thedata stored on the internal data storage devices 110.

The data retrieving device 118 may represent, for example, a floppy diskdrive, a compact disk drive, a magnetic tape drive, or a modem capableof connection to a remote data storage system (e.g., via the internet)etc. In some aspects, the internal data storage device 110 is aremovable computer readable medium such as a floppy disk, a compactdisk, a magnetic tape, etc. containing control logic and/or datarecorded thereon. The computer system 100 may advantageously include orbe programmed by appropriate software for reading the control logicand/or the data from the data storage component once inserted in thedata retrieving device.

The computer system 100 includes a display 120 which is used to displayoutput to a computer user. It should also be noted that the computersystem 100 can be linked to other computer systems 125 a-c in a networkor wide area network to provide centralized access to the computersystem 100.

Software for accessing and processing the nucleotide sequences of anucleic acid sequence of the invention and sequences substantiallyidentical thereto, or a polypeptide sequence of the invention andsequences substantially identical thereto, (such as search tools,compare tools and modeling tools etc.) may reside in main memory 115during execution.

In some aspects, the computer system 100 may further comprise a sequencecomparison algorithm for comparing a nucleic acid sequence of theinvention and sequences substantially identical thereto, or apolypeptide sequence of the invention and sequences substantiallyidentical thereto, stored on a computer readable medium to a referencenucleotide or polypeptide sequence(s) stored on a computer readablemedium. A “sequence comparison algorithm” refers to one or more programswhich are implemented (locally or remotely) on the computer system 100to compare a nucleotide sequence with other nucleotide sequences and/orcompounds stored within a data storage means. For example, the sequencecomparison algorithm may compare the nucleotide sequences of a nucleicacid sequence of the invention and sequences substantially identicalthereto, or a polypeptide sequence of the invention and sequencessubstantially identical thereto, stored on a computer readable medium toreference sequences stored on a computer readable medium to identifyhomologies or structural motifs.

FIG. 2 is a flow diagram illustrating one aspect of a process 200 forcomparing a new nucleotide or protein sequence with a database ofsequences in order to determine the homology levels between the newsequence and the sequences in the database. The database of sequencescan be a private database stored within the computer system 100, or apublic database such as GENBANK that is available through the Internet.

The process 200 begins at a start state 201 and then moves to a state202 wherein the new sequence to be compared is stored to a memory in acomputer system 100. As discussed above, the memory could be any type ofmemory, including RAM or an internal storage device.

The process 200 then moves to a state 204 wherein a database ofsequences is opened for analysis and comparison. The process 200 thenmoves to a state 206 wherein the first sequence stored in the databaseis read into a memory on the computer. A comparison is then performed ata state 210 to determine if the first sequence is the same as the secondsequence. It is important to note that this step is not limited toperforming an exact comparison between the new sequence and the firstsequence in the database. Well-known methods are known to those of skillin the art for comparing two nucleotide or protein sequences, even ifthey are not identical. For example, gaps can be introduced into onesequence in order to raise the homology level between the two testedsequences. The parameters that control whether gaps or other featuresare introduced into a sequence during comparison are normally entered bythe user of the computer system.

Once a comparison of the two sequences has been performed at the state210, a determination is made at a decision state 210 whether the twosequences are the same. Of course, the term “same” is not limited tosequences that are absolutely identical. Sequences that are within thehomology parameters entered by the user will be marked as “same” in theprocess 200.

If a determination is made that the two sequences are the same, theprocess 200 moves to a state 214 wherein the name of the sequence fromthe database is displayed to the user. This state notifies the user thatthe sequence with the displayed name fulfills the homology constraintsthat were entered. Once the name of the stored sequence is displayed tothe user, the process 200 moves to a decision state 218 wherein adetermination is made whether more sequences exist in the database. Ifno more sequences exist in the database, then the process 200 terminatesat an end state 220. However, if more sequences do exist in thedatabase, then the process 200 moves to a state 224 wherein a pointer ismoved to the next sequence in the database so that it can be compared tothe new sequence. In this manner, the new sequence is aligned andcompared with every sequence in the database.

It should be noted that if a determination had been made at the decisionstate 212 that the sequences were not homologous, then the process 200would move immediately to the decision state 218 in order to determineif any other sequences were available in the database for comparison.

Accordingly, one aspect of the invention is a computer system comprisinga processor, a data storage device having stored thereon a nucleic acidsequence of the invention and sequences substantially identical thereto,or a polypeptide sequence of the invention and sequences substantiallyidentical thereto, a data storage device having retrievably storedthereon reference nucleotide sequences or polypeptide sequences to becompared to a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto and a sequencecomparer for conducting the comparison. The sequence comparer mayindicate a homology level between the sequences compared or identifystructural motifs in the above described nucleic acid code of nucleicacid sequences of the invention and sequences substantially identicalthereto, or a polypeptide sequence of the invention and sequencessubstantially identical thereto, or it may identify structural motifs insequences which are compared to these nucleic acid codes and polypeptidecodes. In some aspects, the data storage device may have stored thereonthe sequences of at least 2, 5, 10, 15, 20, 25, 30 or 40 or more of thenucleic acid sequences of the invention and sequences substantiallyidentical thereto, or the polypeptide sequences of the invention andsequences substantially identical thereto.

Another aspect of the invention is a method for determining the level ofhomology between a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto and a referencenucleotide sequence. The method including reading the nucleic acid codeor the polypeptide code and the reference nucleotide or polypeptidesequence through the use of a computer program which determines homologylevels and determining homology between the nucleic acid code orpolypeptide code and the reference nucleotide or polypeptide sequencewith the computer program. The computer program may be any of a numberof computer programs for determining homology levels, including thosespecifically enumerated herein, (e.g., BLAST2N with the defaultparameters or with any modified parameters). The method may beimplemented using the computer systems described above. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30 or 40 ormore of the above described nucleic acid sequences of the invention, orthe polypeptide sequences of the invention through use of the computerprogram and determining homology between the nucleic acid codes orpolypeptide codes and reference nucleotide sequences or polypeptidesequences.

FIG. 3 is a flow diagram illustrating one aspect of a process 250 in acomputer for determining whether two sequences are homologous. Theprocess 250 begins at a start state 252 and then moves to a state 254wherein a first sequence to be compared is stored to a memory. Thesecond sequence to be compared is then stored to a memory at a state256. The process 250 then moves to a state 260 wherein the firstcharacter in the first sequence is read and then to a state 262 whereinthe first character of the second sequence is read. It should beunderstood that if the sequence is a nucleotide sequence, then thecharacter would normally be either A, T, C, G or U. If the sequence is aprotein sequence, then it is preferably in the single letter amino acidcode so that the first and sequence sequences can be easily compared.

A determination is then made at a decision state 264 whether the twocharacters are the same. If they are the same, then the process 250moves to a state 268 wherein the next characters in the first and secondsequences are read. A determination is then made whether the nextcharacters are the same. If they are, then the process 250 continuesthis loop until two characters are not the same. If a determination ismade that the next two characters are not the same, the process 250moves to a decision state 274 to determine whether there are any morecharacters either sequence to read.

If there are not any more characters to read, then the process 250 movesto a state 276 wherein the level of homology between the first andsecond sequences is displayed to the user. The level of homology isdetermined by calculating the proportion of characters between thesequences that were the same out of the total number of sequences in thefirst sequence. Thus, if every character in a first 100 nucleotidesequence aligned with a every character in a second sequence, thehomology level would be 100%.

Alternatively, the computer program may be a computer program whichcompares the nucleotide sequences of a nucleic acid sequence as setforth in the invention, to one or more reference nucleotide sequences inorder to determine whether the nucleic acid code of a nucleic acidsequence of the invention and sequences substantially identical thereto,differs from a reference nucleic acid sequence at one or more positions.In one aspect such a program records the length and identity ofinserted, deleted or substituted nucleotides with respect to thesequence of either the reference polynucleotide or a nucleic acidsequence of the invention and sequences substantially identical thereto.In one aspect, the computer program may be a program which determineswhether a nucleic acid sequence of the invention and sequencessubstantially identical thereto, contains a single nucleotidepolymorphism (SNP) with respect to a reference nucleotide sequence.

Another aspect of the invention is a method for determining whether anucleic acid sequence of the invention and sequences substantiallyidentical thereto, differs at one or more nucleotides from a referencenucleotide sequence comprising the steps of reading the nucleic acidcode and the reference nucleotide sequence through use of a computerprogram which identifies differences between nucleic acid sequences andidentifying differences between the nucleic acid code and the referencenucleotide sequence with the computer program. In some aspects, thecomputer program is a program which identifies single nucleotidepolymorphisms. The method may be implemented by the computer systemsdescribed above and the method illustrated in FIG. 3. The method mayalso be performed by reading at least 2, 5, 10, 15, 20, 25, 30, or 40 ormore of the nucleic acid sequences of the invention and sequencessubstantially identical thereto and the reference nucleotide sequencesthrough the use of the computer program and identifying differencesbetween the nucleic acid codes and the reference nucleotide sequenceswith the computer program.

In other aspects the computer based system may further comprise anidentifier for identifying features within a nucleic acid sequence ofthe invention or a polypeptide sequence of the invention and sequencessubstantially identical thereto.

An “identifier” refers to one or more programs which identifies certainfeatures within a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto. In one aspect,the identifier may comprise a program which identifies an open readingframe in a nucleic acid sequence of the invention and sequencessubstantially identical thereto.

FIG. 4 is a flow diagram illustrating one aspect of an identifierprocess 300 for detecting the presence of a feature in a sequence. Theprocess 300 begins at a start state 302 and then moves to a state 304wherein a first sequence that is to be checked for features is stored toa memory 115 in the computer system 100. The process 300 then moves to astate 306 wherein a database of sequence features is opened. Such adatabase would include a list of each feature's attributes along withthe name of the feature. For example, a feature name could be“Initiation Codon” and the attribute would be “ATG”. Another examplewould be the feature name “TAATAA Box” and the feature attribute wouldbe “TAATAA”. An example of such a database is produced by the Universityof Wisconsin Genetics Computer Group. Alternatively, the features may bestructural polypeptide motifs such as alpha helices, beta sheets, orfunctional polypeptide motifs such as enzymatic active sites,helix-turn-helix motifs or other motifs known to those skilled in theart.

Once the database of features is opened at the state 306, the process300 moves to a state 308 wherein the first feature is read from thedatabase. A comparison of the attribute of the first feature with thefirst sequence is then made at a state 310. A determination is then madeat a decision state 316 whether the attribute of the feature was foundin the first sequence. If the attribute was found, then the process 300moves to a state 318 wherein the name of the found feature is displayedto the user.

The process 300 then moves to a decision state 320 wherein adetermination is made whether move features exist in the database. If nomore features do exist, then the process 300 terminates at an end state324. However, if more features do exist in the database, then theprocess 300 reads the next sequence feature at a state 326 and loopsback to the state 310 wherein the attribute of the next feature iscompared against the first sequence. It should be noted, that if thefeature attribute is not found in the first sequence at the decisionstate 316, the process 300 moves directly to the decision state 320 inorder to determine if any more features exist in the database.

Accordingly, another aspect of the invention is a method of identifyinga feature within a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto, comprisingreading the nucleic acid code(s) or polypeptide code(s) through the useof a computer program which identifies features therein and identifyingfeatures within the nucleic acid code(s) with the computer program. Inone aspect, computer program comprises a computer program whichidentifies open reading frames. The method may be performed by reading asingle sequence or at least 2, 5, 10, 15, 20, 25, 30, or 40 of thenucleic acid sequences of the invention and sequences substantiallyidentical thereto, or the polypeptide sequences of the invention andsequences substantially identical thereto, through the use of thecomputer program and identifying features within the nucleic acid codesor polypeptide codes with the computer program.

A nucleic acid sequence of the invention and sequences substantiallyidentical thereto, or a polypeptide sequence of the invention andsequences substantially identical thereto, may be stored and manipulatedin a variety of data processor programs in a variety of formats. Forexample, a nucleic acid sequence of the invention and sequencessubstantially identical thereto, or a polypeptide sequence of theinvention and sequences substantially identical thereto, may be storedas text in a word processing file, such as Microsoft WORD™ orWORDPERFECT™ or as an ASCII file in a variety of database programsfamiliar to those of skill in the art, such as DB2™, SYBASE™, orORACLE™. In addition, many computer programs and databases may be usedas sequence comparison algorithms, identifiers, or sources of referencenucleotide sequences or polypeptide sequences to be compared to anucleic acid sequence of the invention and sequences substantiallyidentical thereto, or a polypeptide sequence of the invention andsequences substantially identical thereto. The following list isintended not to limit the invention but to provide guidance to programsand databases which are useful with the nucleic acid sequences of theinvention and sequences substantially identical thereto, or thepolypeptide sequences of the invention and sequences substantiallyidentical thereto.

The programs and databases which may be used include, but are notlimited to: MacPattern (EMBL), DiscoveryBase (Molecular ApplicationsGroup), GeneMine (Molecular Applications Group), Look (MolecularApplications Group), MacLook (Molecular Applications Group), BLAST andBLAST2 (NCBI), BLASTN and BLASTX (Altschul et al, J. Mol. Biol. 215:403, 1990), FASTA (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988), FASTDB (Brutlag et al. Comp. App. Biosci. 6:237-245, 1990),Catalyst (Molecular Simulations Inc.), Catalyst/SHAPE (MolecularSimulations Inc.), Cerius².DBAccess (Molecular Simulations Inc.),HypoGen (Molecular Simulations Inc.), Insight II, (Molecular SimulationsInc.), Discover (Molecular Simulations Inc.), CHARMm (MolecularSimulations Inc.), Felix (Molecular Simulations Inc.), DelPhi,(Molecular Simulations Inc.), QuanteMM, (Molecular Simulations Inc.),Homology (Molecular Simulations Inc.), Modeler (Molecular SimulationsInc.), ISIS (Molecular Simulations Inc.), Quanta/Protein Design(Molecular Simulations Inc.), WebLab (Molecular Simulations Inc.),WebLab Diversity Explorer (Molecular Simulations Inc.), Gene Explorer(Molecular Simulations Inc.), SeqFold (Molecular Simulations Inc.), theMDL Available Chemicals Directory database, the MDL Drug Data Reportdata base, the Comprehensive Medicinal Chemistry database, Derwents'sWorld Drug Index database, the BioByteMasterFile database, the Genbankdatabase and the Genseqn database. Many other programs and data baseswould be apparent to one of skill in the art given the presentdisclosure.

Motifs which may be detected using the above programs include sequencesencoding leucine zippers, helix-turn-helix motifs, glycosylation sites,ubiquitination sites, alpha helices and beta sheets, signal sequencesencoding signal peptides which direct the secretion of the encodedproteins, sequences implicated in transcription regulation such ashomeoboxes, acidic stretches, enzymatic active sites, substrate bindingsites and enzymatic cleavage sites.

Hybridization of Nucleic Acids

The invention provides isolated, synthetic or recombinant nucleic acidsthat hybridize under stringent conditions to an exemplary sequence ofthe invention. The stringent conditions can be highly stringentconditions, medium stringent conditions and/or low stringent conditions,including the high and reduced stringency conditions described herein.In one aspect, it is the stringency of the wash conditions that setforth the conditions which determine whether a nucleic acid is withinthe scope of the invention, as discussed below.

In alternative aspects, nucleic acids of the invention as defined bytheir ability to hybridize under stringent conditions can be betweenabout five residues and the full length of nucleic acid of theinvention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50,55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, or more, residues inlength. Nucleic acids shorter than full length are also included. Thesenucleic acids can be useful as, e.g., hybridization probes, labelingprobes, PCR oligonucleotide probes, iRNA (single or double stranded),antisense or sequences encoding antibody binding peptides (epitopes),motifs, active sites and the like.

In one aspect, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprises conditions of about50% formamide at about 37° C. to 42° C. In one aspect, nucleic acids ofthe invention are defined by their ability to hybridize under reducedstringency comprising conditions in about 35% to 25% formamide at about30° C. to 35° C.

Alternatively, nucleic acids of the invention are defined by theirability to hybridize under high stringency comprising conditions at 42°C. in 50% formamide, 5×SSPE, 0.3% SDS, and a repetitive sequenceblocking nucleic acid, such as cot-1 or salmon sperm DNA (e.g., 200ug/ml sheared and denatured salmon sperm DNA). In one aspect, nucleicacids of the invention are defined by their ability to hybridize underreduced stringency conditions comprising 35% formamide at a reducedtemperature of 35° C.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (e.g., GC v. ATcontent) and nucleic acid type (e.g., RNA v. DNA) of the hybridizingregions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

Hybridization may be carried out under conditions of low stringency,moderate stringency or high stringency. As an example of nucleic acidhybridization, a polymer membrane containing immobilized denaturednucleic acids is first prehybridized for 30 minutes at 45° C. in asolution consisting of 0.9 M NaCl, 50 mM NaH₂PO₄, pH 7.0, 5.0 mMNa₂EDTA, 0.5% SDS, 10×Denhardt's and 0.5 mg/ml polyriboadenylic acid.Approximately 2×10⁷ cpm (specific activity 4−9×10⁸ cpm/ug) of ³²Pend-labeled oligonucleotide probe are then added to the solution. After12-16 hours of incubation, the membrane is washed for 30 minutes at roomtemperature in 1×SET (150 mM NaCl, 20 mM Tris hydrochloride, pH 7.8, 1mM Na₂EDTA) containing 0.5% SDS, followed by a 30 minute wash in fresh1×SET at T_(m)−10° C. for the oligonucleotide probe. The membrane isthen exposed to auto-radiographic film for detection of hybridizationsignals.

All of the foregoing hybridizations would be considered to be underconditions of high stringency.

Following hybridization, a filter can be washed to remove anynon-specifically bound detectable probe. The stringency used to wash thefilters can also be varied depending on the nature of the nucleic acidsbeing hybridized, the length of the nucleic acids being hybridized, thedegree of complementarity, the nucleotide sequence composition (e.g., GCv. AT content) and the nucleic acid type (e.g., RNA v. DNA). Examples ofprogressively higher stringency condition washes are as follows: 2×SSC,0.1% SDS at room temperature for 15 minutes (low stringency); 0.1×SSC,0.5% SDS at room temperature for 30 minutes to 1 hour (moderatestringency); 0.1×SSC, 0.5% SDS for 15 to 30 minutes at between thehybridization temperature and 68° C. (high stringency); and 0.15M NaClfor 15 minutes at 72° C. (very high stringency). A final low stringencywash can be conducted in 0.1×SSC at room temperature. The examples aboveare merely illustrative of one set of conditions that can be used towash filters. One of skill in the art would know that there are numerousrecipes for different stringency washes. Some other examples are givenbelow.

Nucleic acids which have hybridized to the probe are identified byautoradiography or other conventional techniques.

The above procedure may be modified to identify nucleic acids havingdecreasing levels of homology to the probe sequence. For example, toobtain nucleic acids of decreasing homology to the detectable probe,less stringent conditions may be used. For example, the hybridizationtemperature may be decreased in increments of 5° C. from 68° C. to 42°C. in a hybridization buffer having a Na+ concentration of approximately1M. Following hybridization, the filter may be washed with 2×SSC, 0.5%SDS at the temperature of hybridization. These conditions are consideredto be “moderate” conditions above 50° C. and “low” conditions below 50°C. A specific example of “moderate” hybridization conditions is when theabove hybridization is conducted at 55° C. A specific example of “lowstringency” hybridization conditions is when the above hybridization isconducted at 45° C.

Alternatively, the hybridization may be carried out in buffers, such as6×SSC, containing formamide at a temperature of 42° C. In this case, theconcentration of formamide in the hybridization buffer may be reduced in5% increments from 50% to 0% to identify clones having decreasing levelsof homology to the probe. Following hybridization, the filter may bewashed with 6×SSC, 0.5% SDS at 50° C. These conditions are considered tobe “moderate” conditions above 25% formamide and “low” conditions below25% formamide. A specific example of “moderate” hybridization conditionsis when the above hybridization is conducted at 30% formamide. Aspecific example of “low stringency” hybridization conditions is whenthe above hybridization is conducted at 10% formamide.

However, the selection of a hybridization format is not critical—it isthe stringency of the wash conditions that set forth the conditionswhich determine whether a nucleic acid is within the scope of theinvention. Wash conditions used to identify nucleic acids within thescope of the invention include, e.g.: a salt concentration of about 0.02molar at pH 7 and a temperature of at least about 50° C. or about 55° C.to about 60° C.; or, a salt concentration of about 0.15 M NaCl at 72° C.for about 15 minutes; or, a salt concentration of about 0.2×SSC at atemperature of at least about 50° C. or about 55° C. to about 60° C. forabout 15 to about 20 minutes; or, the hybridization complex is washedtwice with a solution with a salt concentration of about 2×SSCcontaining 0.1% SDS at room temperature for 15 minutes and then washedtwice by 0.1×SSC containing 0.1% SDS at 68° C. for 15 minutes; or,equivalent conditions. See Sambrook, Tijssen and Ausubel for adescription of SSC buffer and equivalent conditions.

These methods may be used to isolate nucleic acids of the invention. Forexample, the preceding methods may be used to isolate nucleic acidshaving a sequence with at least about 97%, at least 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a nucleic acidsequence selected from the group consisting of one of the sequences ofThe invention and sequences substantially identical thereto, orfragments comprising at least about 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 consecutive bases thereof and thesequences complementary thereto. Homology may be measured using thealignment algorithm. For example, the homologous polynucleotides mayhave a coding sequence which is a naturally occurring allelic variant ofone of the coding sequences described herein. Such allelic variants mayhave a substitution, deletion or addition of one or more nucleotideswhen compared to the nucleic acids of The invention or the sequencescomplementary thereto.

Additionally, the above procedures may be used to isolate nucleic acidswhich encode polypeptides having at least about 99%, 95%, at least 90%,at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, atleast 60%, at least 55%, or at least 50% homology to a polypeptidehaving the sequence of one of amino acid sequences of the invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof as determined using asequence alignment algorithm (e.g., such as the FASTA version 3.0t78algorithm with the default parameters).

Oligonucleotides Probes and Methods for Using them

The invention also provides nucleic acid probes that can be used, e.g.,for identifying nucleic acids encoding a polypeptide with an isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase activity or fragments thereof or for identifying isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase genes. In one aspect, the probe comprises at least 10consecutive bases of a nucleic acid of the invention. Alternatively, aprobe of the invention can be at least about 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50,60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to60 about 30 to 70, consecutive bases of a sequence as set forth in anucleic acid of the invention. The probes identify a nucleic acid bybinding and/or hybridization. The probes can be used in arrays of theinvention, see discussion below, including, e.g., capillary arrays. Theprobes of the invention can also be used to isolate other nucleic acidsor polypeptides.

The isolated nucleic acids of the invention and sequences substantiallyidentical thereto, the sequences complementary thereto, or a fragmentcomprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200,300, 400, or 500 consecutive bases of one of the sequences of Theinvention and sequences substantially identical thereto, or thesequences complementary thereto may also be used as probes to determinewhether a biological sample, such as a soil sample, contains an organismhaving a nucleic acid sequence of the invention or an organism fromwhich the nucleic acid was obtained. In such procedures, a biologicalsample potentially harboring the organism from which the nucleic acidwas isolated is obtained and nucleic acids are obtained from the sample.The nucleic acids are contacted with the probe under conditions whichpermit the probe to specifically hybridize to any complementarysequences from which are present therein.

Where necessary, conditions which permit the probe to specificallyhybridize to complementary sequences may be determined by placing theprobe in contact with complementary sequences from samples known tocontain the complementary sequence as well as control sequences which donot contain the complementary sequence. Hybridization conditions, suchas the salt concentration of the hybridization buffer, the formamideconcentration of the hybridization buffer, or the hybridizationtemperature, may be varied to identify conditions which allow the probeto hybridize specifically to complementary nucleic acids.

If the sample contains the organism from which the nucleic acid wasisolated, specific hybridization of the probe is then detected.Hybridization may be detected by labeling the probe with a detectableagent such as a radioactive isotope, a fluorescent dye or an enzymecapable of catalyzing the formation of a detectable product.

Many methods for using the labeled probes to detect the presence ofcomplementary nucleic acids in a sample are familiar to those skilled inthe art. These include Southern Blots, Northern Blots, colonyhybridization procedures and dot blots. Protocols for each of theseprocedures are provided in Ausubel et al. Current Protocols in MolecularBiology, John Wiley 503 Sons, Inc. (1997) and Sambrook et al., MolecularCloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor LaboratoryPress (1989.

Alternatively, more than one probe (at least one of which is capable ofspecifically hybridizing to any complementary sequences which arepresent in the nucleic acid sample), may be used in an amplificationreaction to determine whether the sample contains an organism containinga nucleic acid sequence of the invention (e.g., an organism from whichthe nucleic acid was isolated). Typically, the probes compriseoligonucleotides. In one aspect, the amplification reaction may comprisea PCR reaction. PCR protocols are described in Ausubel and Sambrook,supra. Alternatively, the amplification may comprise a ligase chainreaction, 3SR, or strand displacement reaction. (See Barany, F., “TheLigase Chain Reaction in a PCR World”, PCR Methods and Applications1:5-16, 1991; E. Fahy et al., “Self-sustained Sequence Replication(3SR): An Isothermal Transcription-based Amplification SystemAlternative to PCR”, PCR Methods and Applications 1:25-33, 1991; andWalker G. T. et al., “Strand Displacement Amplification—an Isothermal invitro DNA Amplification Technique”, Nucleic Acid Research 20:1691-1696,1992). In such procedures, the nucleic acids in the sample are contactedwith the probes, the amplification reaction is performed and anyresulting amplification product is detected. The amplification productmay be detected by performing gel electrophoresis on the reactionproducts and staining the gel with an intercalator such as ethidiumbromide. Alternatively, one or more of the probes may be labeled with aradioactive isotope and the presence of a radioactive amplificationproduct may be detected by autoradiography after gel electrophoresis.

Probes derived from sequences near the ends of the sequences of Theinvention and sequences substantially identical thereto, may also beused in chromosome walking procedures to identify clones containinggenomic sequences located adjacent to the sequences of The invention andsequences substantially identical thereto. Such methods allow theisolation of genes which encode additional proteins from the hostorganism.

The isolated nucleic acids of the invention and sequences substantiallyidentical thereto, the sequences complementary thereto, or a fragmentcomprising at least 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200,300, 400, or 500 consecutive bases of one of the sequences of theinvention and sequences substantially identical thereto, or thesequences complementary thereto may be used as probes to identify andisolate related nucleic acids. In some aspects, the related nucleicacids may be cDNAs or genomic DNAs from organisms other than the onefrom which the nucleic acid was isolated. For example, the otherorganisms may be related organisms. In such procedures, a nucleic acidsample is contacted with the probe under conditions which permit theprobe to specifically hybridize to related sequences. Hybridization ofthe probe to nucleic acids from the related organism is then detectedusing any of the methods described above.

By varying the stringency of the hybridization conditions used toidentify nucleic acids, such as cDNAs or genomic DNAs, which hybridizeto the detectable probe, nucleic acids having different levels ofhomology to the probe can be identified and isolated. Stringency may bevaried by conducting the hybridization at varying temperatures below themelting temperatures of the probes. The melting temperature, T_(m), isthe temperature (under defined ionic strength and pH) at which 50% ofthe target sequence hybridizes to a perfectly complementary probe. Verystringent conditions are selected to be equal to or about 5° C. lowerthan the T_(m) for a particular probe. The melting temperature of theprobe may be calculated using the following formulas:

For probes between 14 and 70 nucleotides in length the meltingtemperature (T_(m)) is calculated using the formula: T_(m)=81.5+16.6(log[Na+])+0.41(fraction G+C)−(600/N) where N is the length of the probe.

If the hybridization is carried out in a solution containing formamide,the melting temperature may be calculated using the equation:T_(m)=81.5+16.6(log [Na+])+0.41(fraction G+C)−(0.63% formamide)−(600/N)where N is the length of the probe.

Prehybridization may be carried out in 6×SSC, 5×Denhardt's reagent, 0.5%SDS, 100 μg/ml denatured fragmented salmon sperm DNA or 6×SSC,5×Denhardt's reagent, 0.5% SDS, 100 μg/ml denatured fragmented salmonsperm DNA, 50% formamide. The formulas for SSC and Denhardt's solutionsare listed in Sambrook et al., supra.

Hybridization is conducted by adding the detectable probe to theprehybridization solutions listed above. Where the probe comprisesdouble stranded DNA, it is denatured before addition to thehybridization solution. The filter is contacted with the hybridizationsolution for a sufficient period of time to allow the probe to hybridizeto cDNAs or genomic DNAs containing sequences complementary thereto orhomologous thereto. For probes over 200 nucleotides in length, thehybridization may be carried out at 15-25° C. below the T_(m). Forshorter probes, such as oligonucleotide probes, the hybridization may beconducted at 5-10° C. below the T_(m). Typically, for hybridizations in6×SSC, the hybridization is conducted at approximately 68° C. Usually,for hybridizations in 50% formamide containing solutions, thehybridization is conducted at approximately 42° C.

Inhibiting Expression of Isomerases

The invention provides nucleic acids complementary to (e.g., antisensesequences to) the nucleic acids of the invention, e.g., an isomerase-,e.g., a racemase-, e.g., an amino acid racemase-, an alanine racemase-,and/or an epimerase-isomerase-, e.g., racemase-, e.g., amino acidracemase-, alanine racemase-, and/or epimerase-encoding nucleic acids.Antisense sequences are capable of inhibiting the transport, splicing ortranscription of an isomerase-, e.g., a racemase-, e.g., an amino acidracemase-, an alanine racemase-, and/or an epimerase-isomerase-, e.g.,racemase-, e.g., amino acid racemase-, alanine racemase-, and/orepimerase-encoding genes. The inhibition can be effected through thetargeting of genomic DNA or messenger RNA. The transcription or functionof targeted nucleic acid can be inhibited, for example, by hybridizationand/or cleavage. One particularly useful set of inhibitors provided bythe present invention includes oligonucleotides which are able to eitherbind an isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase gene or message, in either case preventing orinhibiting the production or function of an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase. Theassociation can be through sequence specific hybridization. Anotheruseful class of inhibitors includes oligonucleotides which causeinactivation or cleavage of an isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase message. Theoligonucleotide can have enzyme activity which causes such cleavage,such as ribozymes. The oligonucleotide can be chemically modified orconjugated to an enzyme or composition capable of cleaving thecomplementary nucleic acid. A pool of many different sucholigonucleotides can be screened for those with the desired activity.Thus, the invention provides various compositions for the inhibition ofisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase expression on a nucleic acid and/or protein level,e.g., antisense, iRNA and ribozymes comprising isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasesequences of the invention and the anti-isomerase, e.g., anti-racemase,e.g., anti-amino acid racemase, anti-alanine racemase, and/oranti-epimerase antibodies of the invention.

The compositions of the invention for the inhibition of isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseexpression (e.g., antisense, iRNA, ribozymes, antibodies) can be used aspharmaceutical compositions.

Antisense Oligonucleotides

The invention provides antisense oligonucleotides capable of binding anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase message which can inhibit, for example, isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase activity by targeting mRNA. Strategies for designing antisenseoligonucleotides are well described in the scientific and patentliterature, and the skilled artisan can design such isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseoligonucleotides using the novel reagents of the invention. For example,gene walking/RNA mapping protocols to screen for effective antisenseoligonucleotides are well known in the art, see, e.g., Ho (2000) MethodsEnzymol. 314:168-183, describing an RNA mapping assay, which is based onstandard molecular techniques to provide an easy and reliable method forpotent antisense sequence selection. See also Smith (2000) Eur. J.Pharm. Sci. 11:191-198.

Naturally occurring nucleic acids are used as antisenseoligonucleotides. The antisense oligonucleotides can be of any length;for example, in alternative aspects, the antisense oligonucleotides arebetween about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40.The optimal length can be determined by routine screening. The antisenseoligonucleotides can be present at any concentration. The optimalconcentration can be determined by routine screening. A wide variety ofsynthetic, non-naturally occurring nucleotide and nucleic acid analoguesare known which can address this potential problem. For example, peptidenucleic acids (PNAs) containing non-ionic backbones, such asN-(2-aminoethyl)glycine units can be used. Antisense oligonucleotideshaving phosphorothioate linkages can also be used, as described in WO97/03211; WO 96/39154; Mata (1997) Toxicol Appl Pharmacol 144:189-197;Antisense Therapeutics, ed. Agrawal (Humana Press, Totowa, N.J., 1996).Antisense oligonucleotides having synthetic DNA backbone analoguesprovided by the invention can also include phosphoro-dithioate,methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate,3′-thioacetal, methylene(methylimino), 3′-N-carbamate, and morpholinocarbamate nucleic acids, as described above.

Combinatorial chemistry methodology can be used to create vast numbersof oligonucleotides that can be rapidly screened for specificoligonucleotides that have appropriate binding affinities andspecificities toward any target, such as the sense and antisenseisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase sequences of the invention (see, e.g., Gold (1995) J.of Biol. Chem. 270:13581-13584).

Inhibitory Ribozymes

The invention provides ribozymes capable of binding an isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasemessage. These ribozymes can inhibit isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase activity by,e.g., targeting mRNA. Strategies for designing ribozymes and selectingthe an isomerase-, e.g., a racemase-, e.g., an amino acid racemase-, analanine racemase-, and/or an epimerase-isomerase-, e.g., racemase-,e.g., amino acid racemase-, alanine racemase-, and/or epimerase-specificantisense sequence for targeting are well described in the scientificand patent literature, and the skilled artisan can design such ribozymesusing the novel reagents (e.g. nucleic acids) of the invention.Ribozymes act by binding to a target RNA through the target RNA bindingportion of a ribozyme which is held in close proximity to an enzymaticportion of the RNA that cleaves the target RNA. Thus, the ribozymerecognizes and binds a target RNA through complementary base-pairing,and once bound to the correct site, acts enzymatically to cleave andinactivate the target RNA. Cleavage of a target RNA in such a mannerwill destroy its ability to direct synthesis of an encoded protein ifthe cleavage occurs in the coding sequence. After a ribozyme has boundand cleaved its RNA target, it can be released from that RNA to bind andcleave new targets repeatedly.

In some circumstances, the enzymatic nature of a ribozyme can beadvantageous over other technologies, such as antisense technology(where a nucleic acid molecule simply binds to a nucleic acid target toblock its transcription, translation or association with anothermolecule) as the effective concentration of ribozyme necessary to effecta therapeutic treatment can be lower than that of an antisenseoligonucleotide. This potential advantage reflects the ability of theribozyme to act enzymatically. Thus, a single ribozyme molecule is ableto cleave many molecules of target RNA. In addition, a ribozyme istypically a highly specific inhibitor, with the specificity ofinhibition depending not only on the base pairing mechanism of binding,but also on the mechanism by which the molecule inhibits the expressionof the RNA to which it binds. That is, the inhibition is caused bycleavage of the RNA target and so specificity is defined as the ratio ofthe rate of cleavage of the targeted RNA over the rate of cleavage ofnon-targeted RNA. This cleavage mechanism is dependent upon factorsadditional to those involved in base pairing. Thus, the specificity ofaction of a ribozyme can be greater than that of antisenseoligonucleotide binding the same RNA site.

The ribozyme of the invention, e.g., an enzymatic ribozyme RNA molecule,can be formed in a hammerhead motif, a hairpin motif, as a hepatitisdelta virus motif, a group I intron motif and/or an RNaseP-like RNA inassociation with an RNA guide sequence. Examples of hammerhead motifsare described by, e.g., Rossi (1992) Aids Research and HumanRetroviruses 8:183; hairpin motifs by Hampel (1989) Biochemistry28:4929, and Hampel (1990) Nuc. Acids Res. 18:299; the hepatitis deltavirus motif by Perrotta (1992) Biochemistry 31:16; the RNaseP motif byGuerrier-Takada (1983) Cell 35:849; and the group I intron by Cech U.S.Pat. No. 4,987,071. The recitation of these specific motifs is notintended to be limiting. Those skilled in the art will recognize that aribozyme of the invention, e.g., an enzymatic RNA molecule of thisinvention, can have a specific substrate binding site complementary toone or more of the target gene RNA regions. A ribozyme of the inventioncan have a nucleotide sequence within or surrounding that substratebinding site which imparts an RNA cleaving activity to the molecule.

RNA Interference (RNAi)

In one aspect, the invention provides an RNA inhibitory molecule, aso-called “RNAi” molecule, comprising an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase enzymesequence of the invention. The RNAi molecule can comprise adouble-stranded RNA (dsRNA) molecule, e.g., siRNA, miRNA and/or shorthairpin RNA (shRNA) molecules. The RNAi molecule, e.g., siRNA (smallinhibitory RNA) can inhibit expression of an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase enzymegene, and/or miRNA (micro RNA) to inhibit translation of an isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase message. In one aspect, the RNAi molecule, e.g., siRNA and/ormiRNA, is about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or more duplex nucleotides in length. While theinvention is not limited by any particular mechanism of action, the RNAican enter a cell and cause the degradation of a single-stranded RNA(ssRNA) of similar or identical sequences, including endogenous mRNAs.When a cell is exposed to double-stranded RNA (dsRNA), mRNA from thehomologous gene is selectively degraded by a process called RNAinterference (RNAi). A possible basic mechanism behind RNAi is thebreaking of a double-stranded RNA (dsRNA) matching a specific genesequence into short pieces called short interfering RNA, which triggerthe degradation of mRNA that matches its sequence. In one aspect, theRNAi's of the invention are used in gene-silencing therapeutics, see,e.g., Shuey (2002) Drug Discov. Today 7:1040-1046. In one aspect, theinvention provides methods to selectively degrade RNA using the RNAi'smolecules, e.g., siRNA and/or miRNA, of the invention. The process maybe practiced in vitro, ex vivo or in vivo. In one aspect, the RNAimolecules of the invention can be used to generate a loss-of-functionmutation in a cell, an organ or an animal.

In one aspect, intracellular introduction of the RNAi is byinternalization of a target cell specific ligand bonded to an RNAbinding protein comprising an RNAi (e.g., microRNA) is adsorbed. Theligand is specific to a unique target cell surface antigen. The ligandcan be spontaneously internalized after binding to the cell surfaceantigen. If the unique cell surface antigen is not naturallyinternalized after binding to its ligand, internalization can bepromoted by the incorporation of an arginine-rich peptide, or othermembrane permeable peptide, into the structure of the ligand or RNAbinding protein or attachment of such a peptide to the ligand or RNAbinding protein. See, e.g., U.S. Patent App. Pub. Nos. 20060030003;20060025361; 20060019286; 20060019258. In one aspect, the inventionprovides lipid-based formulations for delivering, e.g., introducingnucleic acids of the invention as nucleic acid-lipid particlescomprising an RNAi molecule to a cell, see e.g., U.S. Patent App. Pub.No. 20060008910.

Methods for making and using RNAi molecules, e.g., siRNA and/or miRNA,for selectively degrade RNA are well known in the art, see, e.g., U.S.Pat. Nos. 6,506,559; 6,511,824; 6,515,109; 6,489,127.

Modification of Nucleic Acids

The invention provides methods of generating variants of the nucleicacids of the invention, e.g., those encoding an isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerase.These methods can be repeated or used in various combinations togenerate isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases having an altered or differentactivity or an altered or different stability from that of an isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase encoded by the template nucleic acid. These methods also canbe repeated or used in various combinations, e.g., to generatevariations in gene/message expression, message translation or messagestability. In another aspect, the genetic composition of a cell isaltered by, e.g., modification of a homologous gene ex vivo, followed byits reinsertion into the cell.

A nucleic acid of the invention can be altered by any means. Forexample, random or stochastic methods, or, non-stochastic, or “directedevolution,” methods, see, e.g., U.S. Pat. No. 6,361,974. Methods forrandom mutation of genes are well known in the art, see, e.g., U.S. Pat.No. 5,830,696. For example, mutagens can be used to randomly mutate agene. Mutagens include, e.g., ultraviolet light or gamma irradiation, ora chemical mutagen, e.g., mitomycin, nitrous acid, photoactivatedpsoralens, alone or in combination, to induce DNA breaks amenable torepair by recombination. Other chemical mutagens include, for example,sodium bisulfite, nitrous acid, hydroxylamine, hydrazine or formic acid.Other mutagens are analogues of nucleotide precursors, e.g.,nitrosoguanidine, 5-bromouracil, 2-aminopurine, or acridine. Theseagents can be added to a PCR reaction in place of the nucleotideprecursor thereby mutating the sequence. Intercalating agents such asproflavine, acriflavine, quinacrine and the like can also be used.

Any technique in molecular biology can be used, e.g., random PCRmutagenesis, see, e.g., Rice (1992) Proc. Natl. Acad. Sci. USA89:5467-5471; or, combinatorial multiple cassette mutagenesis, see,e.g., Crameri (1995) Biotechniques 18:194-196. Alternatively, nucleicacids, e.g., genes, can be reassembled after random, or “stochastic,”fragmentation, see, e.g., U.S. Pat. Nos. 6,291,242; 6,287,862;6,287,861; 5,955,358; 5,830,721; 5,824,514; 5,811,238; 5,605,793. Inalternative aspects, modifications, additions or deletions areintroduced by error-prone PCR, shuffling, oligonucleotide-directedmutagenesis, assembly PCR, sexual PCR mutagenesis, in vivo mutagenesis,cassette mutagenesis, recursive ensemble mutagenesis, exponentialensemble mutagenesis, site-specific mutagenesis, gene reassembly (e.g.,GeneReassembly, see, e.g., U.S. Pat. No. 6,537,776), gene sitesaturation mutagenesis (GSSM), synthetic ligation reassembly (SLR),recombination, recursive sequence recombination, phosphothioate-modifiedDNA mutagenesis, uracil-containing template mutagenesis, gapped duplexmutagenesis, point mismatch repair mutagenesis, repair-deficient hoststrain mutagenesis, chemical mutagenesis, radiogenic mutagenesis,deletion mutagenesis, restriction-selection mutagenesis,restriction-purification mutagenesis, artificial gene synthesis,ensemble mutagenesis, chimeric nucleic acid multimer creation, and/or acombination of these and other methods.

The following publications describe a variety of recursive recombinationprocedures and/or methods which can be incorporated into the methods ofthe invention: Stemmer (1999) “Molecular breeding of viruses fortargeting and other clinical properties” Tumor Targeting 4:1-4; Ness(1999) Nature Biotechnology 17:893-896; Chang (1999) “Evolution of acytokine using DNA family shuffling” Nature Biotechnology 17:793-797;Minshull (1999) “Protein evolution by molecular breeding” CurrentOpinion in Chemical Biology 3:284-290; Christians (1999) “Directedevolution of thymidine kinase for AZT phosphorylation using DNA familyshuffling” Nature Biotechnology 17:259-264; Crameri (1998) “DNAshuffling of a family of genes from diverse species accelerates directedevolution” Nature 391:288-291; Crameri (1997) “Molecular evolution of anarsenate detoxification pathway by DNA shuffling,” Nature Biotechnology15:436-438; Zhang (1997) “Directed evolution of an effective fucosidasefrom a galactosidase by DNA shuffling and screening” Proc. Natl. Acad.Sci. USA 94:4504-4509; Patten et al. (1997) “Applications of DNAShuffling to Pharmaceuticals and Vaccines” Current Opinion inBiotechnology 8:724-733; Crameri et al. (1996) “Construction andevolution of antibody-phage libraries by DNA shuffling” Nature Medicine2:100-103; Gates et al. (1996) “Affinity selective isolation of ligandsfrom peptide libraries through display on a lac repressor ‘headpiecedimer’” Journal of Molecular Biology 255:373-386; Stemmer (1996) “SexualPCR and Assembly PCR” In: The Encyclopedia of Molecular Biology. VCHPublishers, New York. pp. 447-457; Crameri and Stemmer (1995)“Combinatorial multiple cassette mutagenesis creates all thepermutations of mutant and wildtype cassettes” BioTechniques 18:194-195;Stemmer et al. (1995) “Single-step assembly of a gene and entire plasmidform large numbers of oligodeoxyribonucleotides” Gene, 164:49-53;Stemmer (1995) “The Evolution of Molecular Computation” Science 270:1510; Stemmer (1995) “Searching Sequence Space” Bio/Technology13:549-553; Stemmer (1994) “Rapid evolution of a protein in vitro by DNAshuffling” Nature 370:389-391; and Stemmer (1994) “DNA shuffling byrandom fragmentation and reassembly: In vitro recombination formolecular evolution.” Proc. Natl. Acad. Sci. USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling et al. (1997) “Approaches to DNAmutagenesis: an overview” Anal Biochem. 254(2): 157-178; Dale et al.(1996) “Oligonucleotide-directed random mutagenesis using thephosphorothioate method” Methods Mol. Biol. 57:369-374; Smith (1985) “Invitro mutagenesis” Ann. Rev. Genet. 19:423-462; Botstein & Shortle(1985) “Strategies and applications of in vitro mutagenesis” Science229:1193-1201; Carter (1986) “Site-directed mutagenesis” Biochem. J.237:1-7; and Kunkel (1987) “The efficiency of oligonucleotide directedmutagenesis” in Nucleic Acids & Molecular Biology (Eckstein, F. andLilley, D. M. J. eds., Springer Verlag, Berlin)); mutagenesis usinguracil containing templates (Kunkel (1985) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Proc. Natl.Acad. Sci. USA 82:488-492; Kunkel et al. (1987) “Rapid and efficientsite-specific mutagenesis without phenotypic selection” Methods inEnzymol. 154, 367-382; and Bass et al. (1988) “Mutant Trp repressorswith new DNA-binding specificities” Science 242:240-245);oligonucleotide-directed mutagenesis (Methods in Enzymol. 100: 468-500(1983); Methods in Enzymol. 154: 329-350 (1987); Zoller (1982)“Oligonucleotide-directed mutagenesis using M13-derived vectors: anefficient and general procedure for the production of point mutations inany DNA fragment” Nucleic Acids Res. 10:6487-6500; Zoller & Smith (1983)“Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13vectors” Methods in Enzymol. 100:468-500; and Zoller (1987)Oligonucleotide-directed mutagenesis: a simple method using twooligonucleotide primers and a single-stranded DNA template” Methods inEnzymol. 154:329-350); phosphorothioate-modified DNA mutagenesis (Taylor(1985) “The use of phosphorothioate-modified DNA in restriction enzymereactions to prepare nicked DNA” Nucl. Acids Res. 13: 8749-8764; Taylor(1985) “The rapid generation of oligonucleotide-directed mutations athigh frequency using phosphorothioate-modified DNA” Nucl. Acids Res. 13:8765-8787 (1985); Nakamaye (1986) “Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and itsapplication to oligonucleotide-directed mutagenesis” Nucl. Acids Res.14: 9679-9698; Sayers (1988) “Y-T Exonucleases in phosphorothioate-basedoligonucleotide-directed mutagenesis” Nucl. Acids Res. 16:791-802; andSayers et al. (1988) “Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restrictionendonucleases in the presence of ethidium bromide” Nucl. Acids Res. 16:803-814); mutagenesis using gapped duplex DNA (Kramer et al. (1984) “Thegapped duplex DNA approach to oligonucleotide-directed mutationconstruction” Nucl. Acids Res. 12: 9441-9456; Kramer & Fritz (1987)Methods in Enzymol. “Oligonucleotide-directed construction of mutationsvia gapped duplex DNA” 154:350-367; Kramer (1988) “Improved enzymatic invitro reactions in the gapped duplex DNA approach tooligonucleotide-directed construction of mutations” Nucl. Acids Res. 16:7207; and Fritz (1988) “Oligonucleotide-directed construction ofmutations: a gapped duplex DNA procedure without enzymatic reactions invitro” Nucl. Acids Res. 16: 6987-6999).

Additional protocols that can be used to practice the invention includepoint mismatch repair (Kramer (1984) “Point Mismatch Repair” Cell38:879-887), mutagenesis using repair-deficient host strains (Carter etal. (1985) “Improved oligonucleotide site-directed mutagenesis using M13vectors” Nucl. Acids Res. 13: 4431-4443; and Carter (1987) “Improvedoligonucleotide-directed mutagenesis using M13 vectors” Methods inEnzymol. 154: 382-403), deletion mutagenesis (Eghtedarzadeh (1986) “Useof oligonucleotides to generate large deletions” Nucl. Acids Res. 14:5115), restriction-selection and restriction-selection andrestriction-purification (Wells et al. (1986) “Importance ofhydrogen-bond formation in stabilizing the transition state ofsubtilisin” Phil. Trans. R. Soc. Lond. A 317: 415-423), mutagenesis bytotal gene synthesis (Nambiar et al. (1984) “Total synthesis and cloningof a gene coding for the ribonuclease S protein” Science 223: 1299-1301;Sakamar and Khorana (1988) “Total synthesis and expression of a gene forthe a-subunit of bovine rod outer segment guanine nucleotide-bindingprotein (transducin)” Nucl. Acids Res. 14: 6361-6372; Wells et al.(1985) “Cassette mutagenesis: an efficient method for generation ofmultiple mutations at defined sites” Gene 34:315-323; and Grundstrom etal. (1985) “Oligonucleotide-directed mutagenesis by microscale‘shot-gun’ gene synthesis” Nucl. Acids Res. 13: 3305-3316),double-strand break repair (Mandecki (1986); Arnold (1993) “Proteinengineering for unusual environments” Current Opinion in Biotechnology4:450-455. “Oligonucleotide-directed double-strand break repair inplasmids of Escherichia coli: a method for site-specific mutagenesis”Proc. Natl. Acad. Sci. USA, 83:7177-7181). Additional details on many ofthe above methods can be found in Methods in Enzymology Volume 154,which also describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Protocols that can be used to practice the invention are described,e.g., in U.S. Pat. No. 5,605,793 to Stemmer (Feb. 25, 1997), “Methodsfor In Vitro Recombination;” U.S. Pat. No. 5,811,238 to Stemmer et al.(Sep. 22, 1998) “Methods for Generating Polynucleotides having DesiredCharacteristics by Iterative Selection and Recombination;” U.S. Pat. No.5,830,721 to Stemmer et al. (Nov. 3, 1998), “DNA Mutagenesis by RandomFragmentation and Reassembly;” U.S. Pat. No. 5,834,252 to Stemmer, etal. (Nov. 10, 1998) “End-Complementary Polymerase Reaction;” U.S. Pat.No. 5,837,458 to Minshull, et al. (Nov. 17, 1998), “Methods andCompositions for Cellular and Metabolic Engineering;” WO 95/22625,Stemmer and Crameri, “Mutagenesis by Random Fragmentation andReassembly;” WO 96/33207 by Stemmer and Lipschutz “End ComplementaryPolymerase Chain Reaction;” WO 97/20078 by Stemmer and Crameri “Methodsfor Generating Polynucleotides having Desired Characteristics byIterative Selection and Recombination;” WO 97/35966 by Minshull andStemmer, “Methods and Compositions for Cellular and MetabolicEngineering;” WO 99/41402 by Punnonen et al. “Targeting of GeneticVaccine Vectors;” WO 99/41383 by Punnonen et al. “Antigen LibraryImmunization;” WO 99/41369 by Punnonen et al. “Genetic Vaccine VectorEngineering;” WO 99/41368 by Punnonen et al. “Optimization ofImmunomodulatory Properties of Genetic Vaccines;” EP 752008 by Stemmerand Crameri, “DNA Mutagenesis by Random Fragmentation and Reassembly;”EP 0932670 by Stemmer “Evolving Cellular DNA Uptake by RecursiveSequence Recombination;” WO 99/23107 by Stemmer et al., “Modification ofVirus Tropism and Host Range by Viral Genome Shuffling;” WO 99/21979 byApt et al., “Human Papillomavirus Vectors;” WO 98/31837 by del Cardayreet al. “Evolution of Whole Cells and Organisms by Recursive SequenceRecombination;” WO 98/27230 by Patten and Stemmer, “Methods andCompositions for Polypeptide Engineering;” WO 98/27230 by Stemmer etal., “Methods for Optimization of Gene Therapy by Recursive SequenceShuffling and Selection,” WO 00/00632, “Methods for Generating HighlyDiverse Libraries,” WO 00/09679, “Methods for Obtaining in VitroRecombined Polynucleotide Sequence Banks and Resulting Sequences,” WO98/42832 by Arnold et al., “Recombination of Polynucleotide SequencesUsing Random or Defined Primers,” WO 99/29902 by Arnold et al., “Methodfor Creating Polynucleotide and Polypeptide Sequences,” WO 98/41653 byVind, “An in Vitro Method for Construction of a DNA Library,” WO98/41622 by Borchert et al., “Method for Constructing a Library UsingDNA Shuffling,” and WO 98/42727 by Pati and Zarling, “SequenceAlterations using Homologous Recombination.”

Protocols that can be used to practice the invention (providing detailsregarding various diversity generating methods) are described, e.g., inU.S. patent application Ser. No. 09/407,800, “SHUFFLING OF CODON ALTEREDGENES” by Patten et al. filed Sep. 28, 1999; “EVOLUTION OF WHOLE CELLSAND ORGANISMS BY RECURSIVE SEQUENCE RECOMBINATION” by del Cardayre etal., U.S. Pat. No. 6,379,964; “OLIGONUCLEOTIDE MEDIATED NUCLEIC ACIDRECOMBINATION” by Crameri et al., U.S. Pat. Nos. 6,319,714; 6,368,861;6,376,246; 6,423,542; 6,426,224 and PCT/US00/01203; “USE OF CODON-VARIEDOLIGONUCLEOTIDE SYNTHESIS FOR SYNTHETIC SHUFFLING” by Welch et al., U.S.Pat. No. 6,436,675; “METHODS FOR MAKING CHARACTER STRINGS,POLYNUCLEOTIDES & POLYPEPTIDES HAVING DESIRED CHARACTERISTICS” bySelifonov et al., filed Jan. 18, 2000, (PCT/US00/01202) and, e.g.“METHODS FOR MAKING CHARACTER STRINGS, POLYNUCLEOTIDES & POLYPEPTIDESHAVING DESIRED CHARACTERISTICS” by Selifonov et al., filed Jul. 18, 2000(U.S. Ser. No. 09/618,579); “METHODS OF POPULATING DATA STRUCTURES FORUSE IN EVOLUTIONARY SIMULATIONS” by Selifonov and Stemmer, filed Jan.18, 2000 (PCT/US00/01138); and “SINGLE-STRANDED NUCLEIC ACIDTEMPLATE-MEDIATED RECOMBINATION AND NUCLEIC ACID FRAGMENT ISOLATION” byAffholter, filed Sep. 6, 2000 (U.S. Ser. No. 09/656,549); and U.S. Pat.Nos. 6,177,263; 6,153,410.

Non-stochastic, or “directed evolution,” methods include, e.g.,saturation mutagenesis (GSSM), synthetic ligation reassembly (SLR), or acombination thereof are used to modify the nucleic acids of theinvention to generate isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases with new or alteredproperties (e.g., activity under highly acidic or alkaline conditions,high or low temperatures, and the like). Polypeptides encoded by themodified nucleic acids can be screened for an activity. Any testingmodality or protocol can be used, e.g., using a capillary arrayplatform. See, e.g., U.S. Pat. Nos. 6,361,974; 6,280,926; 5,939,250.

Gene Site Saturation Mutagenesis, or, GSSM

The invention also provides methods for making enzyme using Gene SiteSaturation mutagenesis, or, GSSM, as described herein, and also in U.S.Pat. Nos. 6,171,820 and 6,579,258. In one aspect, codon primerscontaining a degenerate N,N,G/T sequence are used to introduce pointmutations into a polynucleotide, e.g., an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase or anantibody of the invention, so as to generate a set of progenypolypeptides in which a full range of single amino acid substitutions isrepresented at each amino acid position, e.g., an amino acid residue inan enzyme active site or ligand binding site targeted to be modified.These oligonucleotides can comprise a contiguous first homologoussequence, a degenerate N,N,G/T sequence, and, in one aspect, a secondhomologous sequence. The downstream progeny translational products fromthe use of such oligonucleotides include all possible amino acid changesat each amino acid site along the polypeptide, because the degeneracy ofthe N,N,G/T sequence includes codons for all 20 amino acids. In oneaspect, one such degenerate oligonucleotide (comprised of, e.g., onedegenerate N,N,G/T cassette) is used for subjecting each original codonin a parental polynucleotide template to a full range of codonsubstitutions. In another aspect, at least two degenerate cassettes areused—either in the same oligonucleotide or not, for subjecting at leasttwo original codons in a parental polynucleotide template to a fullrange of codon substitutions. For example, more than one N,N,G/Tsequence can be contained in one oligonucleotide to introduce amino acidmutations at more than one site. This plurality of N,N,G/T sequences canbe directly contiguous, or separated by one or more additionalnucleotide sequence(s). In another aspect, oligonucleotides serviceablefor introducing additions and deletions can be used either alone or incombination with the codons containing an N,N,G/T sequence, to introduceany combination or permutation of amino acid additions, deletions,and/or substitutions.

In one aspect, simultaneous mutagenesis of two or more contiguous aminoacid positions is done using an oligonucleotide that contains contiguousN,N,G/T triplets, i.e. a degenerate (N,N,G/T)n sequence. In anotheraspect, degenerate cassettes having less degeneracy than the N,N,G/Tsequence are used. For example, it may be desirable in some instances touse (e.g. in an oligonucleotide) a degenerate triplet sequence comprisedof only one N, where said N can be in the first second or third positionof the triplet. Any other bases including any combinations andpermutations thereof can be used in the remaining two positions of thetriplet. Alternatively, it may be desirable in some instances to use(e.g. in an oligo) a degenerate N,N,N triplet sequence.

In one aspect, use of degenerate triplets (e.g., N,N,G/T triplets)allows for systematic and easy generation of a full range of possiblenatural amino acids (for a total of 20 amino acids) into each and everyamino acid position in a polypeptide (in alternative aspects, themethods also include generation of less than all possible substitutionsper amino acid residue, or codon, position). For example, for a 100amino acid polypeptide, 2000 distinct species (i.e. 20 possible aminoacids per position×100 amino acid positions) can be generated. Throughthe use of an oligonucleotide or set of oligonucleotides containing adegenerate N,N,G/T triplet, 32 individual sequences can code for all 20possible natural amino acids. Thus, in a reaction vessel in which aparental polynucleotide sequence is subjected to saturation mutagenesisusing at least one such oligonucleotide, there are generated 32 distinctprogeny polynucleotides encoding 20 distinct polypeptides. In contrast,the use of a non-degenerate oligonucleotide in site-directed mutagenesisleads to only one progeny polypeptide product per reaction vessel.Nondegenerate oligonucleotides can in one aspect be used in combinationwith degenerate primers disclosed; for example, nondegenerateoligonucleotides can be used to generate specific point mutations in aworking polynucleotide. This provides one means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes, and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

In one aspect, each saturation mutagenesis reaction vessel containspolynucleotides encoding at least 20 progeny polypeptide (e.g.,isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases) molecules such that all 20 natural aminoacids are represented at the one specific amino acid positioncorresponding to the codon position mutagenized in the parentalpolynucleotide (other aspects use less than all 20 naturalcombinations). The 32-fold degenerate progeny polypeptides generatedfrom each saturation mutagenesis reaction vessel can be subjected toclonal amplification (e.g. cloned into a suitable host, e.g., E. colihost, using, e.g., an expression vector) and subjected to expressionscreening. When an individual progeny polypeptide is identified byscreening to display a favorable change in property (when compared tothe parental polypeptide, such as increased isomerase activity, e.g.,racemase activity, e.g., amino acid racemase activity, alanine racemaseactivity, and/or epimerase activity under alkaline or acidicconditions), it can be sequenced to identify the correspondinglyfavorable amino acid substitution contained therein.

In one aspect, upon mutagenizing each and every amino acid position in aparental polypeptide using saturation mutagenesis as disclosed herein,favorable amino acid changes may be identified at more than one aminoacid position. One or more new progeny molecules can be generated thatcontain a combination of all or part of these favorable amino acidsubstitutions. For example, if 2 specific favorable amino acid changesare identified in each of 3 amino acid positions in a polypeptide, thepermutations include 3 possibilities at each position (no change fromthe original amino acid, and each of two favorable changes) and 3positions. Thus, there are 3×3×3 or 27 total possibilities, including 7that were previously examined-6 single point mutations (i.e. 2 at eachof three positions) and no change at any position.

In yet another aspect, site-saturation mutagenesis can be used togetherwith shuffling, chimerization, recombination and other mutagenizingprocesses, along with screening. This invention provides for the use ofany mutagenizing process(es), including saturation mutagenesis, in aniterative manner. In one exemplification, the iterative use of anymutagenizing process(es) is used in combination with screening.

The invention also provides for the use of proprietary codon primers(containing a degenerate N,N,N sequence) to introduce point mutationsinto a polynucleotide, so as to generate a set of progeny polypeptidesin which a full range of single amino acid substitutions is representedat each amino acid position (gene site saturation mutagenesis (GSSM)).The oligos used are comprised contiguously of a first homologoussequence, a degenerate N,N,N sequence and preferably but not necessarilya second homologous sequence. The downstream progeny translationalproducts from the use of such oligos include all possible amino acidchanges at each amino acid site along the polypeptide, because thedegeneracy of the N,N,N sequence includes codons for all 20 amino acids.

In one aspect, one such degenerate oligo (comprised of one degenerateN,N,N cassette) is used for subjecting each original codon in a parentalpolynucleotide template to a full range of codon substitutions. Inanother aspect, at least two degenerate N,N,N cassettes are used—eitherin the same oligo or not, for subjecting at least two original codons ina parental polynucleotide template to a full range of codonsubstitutions. Thus, more than one N,N,N sequence can be contained inone oligo to introduce amino acid mutations at more than one site. Thisplurality of N,N,N sequences can be directly contiguous, or separated byone or more additional nucleotide sequence(s). In another aspect, oligosserviceable for introducing additions and deletions can be used eitheralone or in combination with the codons containing an N,N,N sequence, tointroduce any combination or permutation of amino acid additions,deletions and/or substitutions.

In a particular exemplification, it is possible to simultaneouslymutagenize two or more contiguous amino acid positions using an oligothat contains contiguous N,N,N triplets, i.e. a degenerate (N,N,N)_(n)sequence.

In another aspect, the present invention provides for the use ofdegenerate cassettes having less degeneracy than the N,N,N sequence. Forexample, it may be desirable in some instances to use (e.g. in an oligo)a degenerate triplet sequence comprised of only one N, where the N canbe in the first second or third position of the triplet. Any other basesincluding any combinations and permutations thereof can be used in theremaining two positions of the triplet. Alternatively, it may bedesirable in some instances to use (e.g., in an oligo) a degenerateN,N,N triplet sequence, N,N,G/T, or an N,N, G/C triplet sequence.

It is appreciated, however, that the use of a degenerate triplet (suchas N,N,G/T or an N,N, G/C triplet sequence) as disclosed in the instantinvention is advantageous for several reasons. In one aspect, thisinvention provides a means to systematically and fairly easily generatethe substitution of the full range of possible amino acids (for a totalof 20 amino acids) into each and every amino acid position in apolypeptide. Thus, for a 100 amino acid polypeptide, the inventionprovides a way to systematically and fairly easily generate 2000distinct species (i.e., 20 possible amino acids per position times 100amino acid positions). It is appreciated that there is provided, throughthe use of an oligo containing a degenerate N,N,G/T or an N,N, G/Ctriplet sequence, 32 individual sequences that code for 20 possibleamino acids. Thus, in a reaction vessel in which a parentalpolynucleotide sequence is subjected to saturation mutagenesis using onesuch oligo, there are generated 32 distinct progeny polynucleotidesencoding 20 distinct polypeptides. In contrast, the use of anon-degenerate oligo in site-directed mutagenesis leads to only oneprogeny polypeptide product per reaction vessel.

This invention also provides for the use of nondegenerate oligos, whichcan in one aspect be used in combination with degenerate primersdisclosed. It is appreciated that in some situations, it is advantageousto use nondegenerate oligos to generate specific point mutations in aworking polynucleotide. This provides a means to generate specificsilent point mutations, point mutations leading to corresponding aminoacid changes and point mutations that cause the generation of stopcodons and the corresponding expression of polypeptide fragments.

Thus, in one aspect of this invention, each saturation mutagenesisreaction vessel contains polynucleotides encoding at least 20 progenypolypeptide molecules such that all 20 amino acids are represented atthe one specific amino acid position corresponding to the codon positionmutagenized in the parental polynucleotide. The 32-fold degenerateprogeny polypeptides generated from each saturation mutagenesis reactionvessel can be subjected to clonal amplification (e.g., cloned into asuitable E. coli host using an expression vector) and subjected toexpression screening. When an individual progeny polypeptide isidentified by screening to display a favorable change in property (whencompared to the parental polypeptide), it can be sequenced to identifythe correspondingly favorable amino acid substitution contained therein.

It is appreciated that upon mutagenizing each and every amino acidposition in a parental polypeptide using saturation mutagenesis asdisclosed herein, favorable amino acid changes may be identified at morethan one amino acid position. One or more new progeny molecules can begenerated that contain a combination of all or part of these favorableamino acid substitutions. For example, if 2 specific favorable aminoacid changes are identified in each of 3 amino acid positions in apolypeptide, the permutations include 3 possibilities at each position(no change from the original amino acid and each of two favorablechanges) and 3 positions. Thus, there are 3×3×3 or 27 totalpossibilities, including 7 that were previously examined-6 single pointmutations (i.e., 2 at each of three positions) and no change at anyposition.

Thus, in a non-limiting exemplification, this invention provides for theuse of saturation mutagenesis in combination with additionalmutagenization processes, such as process where two or more relatedpolynucleotides are introduced into a suitable host cell such that ahybrid polynucleotide is generated by recombination and reductivereassortment.

In addition to performing mutagenesis along the entire sequence of agene, the instant invention provides that mutagenesis can be use toreplace each of any number of bases in a polynucleotide sequence,wherein the number of bases to be mutagenized is preferably everyinteger from 15 to 100,000. Thus, instead of mutagenizing every positionalong a molecule, one can subject every or a discrete number of bases(preferably a subset totaling from 15 to 100,000) to mutagenesis.Preferably, a separate nucleotide is used for mutagenizing each positionor group of positions along a polynucleotide sequence. A group of 3positions to be mutagenized may be a codon. The mutations are preferablyintroduced using a mutagenic primer, containing a heterologous cassette,also referred to as a mutagenic cassette. Exemplary cassettes can havefrom 1 to 500 bases. Each nucleotide position in such heterologouscassettes be N, A, C, G, T, A/C, A/G, A/T, C/G, C/T, G/T, C/G/T, A/G/T,A/C/T, A/C/G, or E, where E is any base that is not A, C, G, or T (E canbe referred to as a designer oligo).

In a general sense, saturation mutagenesis is comprised of mutagenizinga complete set of mutagenic cassettes (wherein each cassette ispreferably about 1-500 bases in length) in defined polynucleotidesequence to be mutagenized (wherein the sequence to be mutagenized ispreferably from about 15 to 100,000 bases in length). Thus, a group ofmutations (ranging from 1 to 100 mutations) is introduced into eachcassette to be mutagenized. A grouping of mutations to be introducedinto one cassette can be different or the same from a second grouping ofmutations to be introduced into a second cassette during the applicationof one round of saturation mutagenesis. Such groupings are exemplifiedby deletions, additions, groupings of particular codons and groupings ofparticular nucleotide cassettes.

Defined sequences to be mutagenized include a whole gene, pathway, cDNA,an entire open reading frame (ORF) and entire promoter, enhancer,repressor/transactivator, origin of replication, intron, operator, orany polynucleotide functional group. Generally, a “defined sequences”for this purpose may be any polynucleotide that a 15 base-polynucleotidesequence and polynucleotide sequences of lengths between 15 bases and15,000 bases (this invention specifically names every integer inbetween). Considerations in choosing groupings of codons include typesof amino acids encoded by a degenerate mutagenic cassette.

In one exemplification a grouping of mutations that can be introducedinto a mutagenic cassette, this invention specifically provides fordegenerate codon substitutions (using degenerate oligos) that code for2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 and 20amino acids at each position and a library of polypeptides encodedthereby.

Synthetic Ligation Reassembly (SLR)

The invention provides a non-stochastic gene modification system termed“synthetic ligation reassembly,” or simply “SLR,” a “directed evolutionprocess,” to generate polypeptides, e.g., isomerases, e.g., racemases,e.g., amino acid racemases, alanine racemases, and/or epimerases, orantibodies of the invention, with new or altered properties.

SLR is a method of ligating oligonucleotide fragments togethernon-stochastically. This method differs from stochastic oligonucleotideshuffling in that the nucleic acid building blocks are not shuffled,concatenated or chimerized randomly, but rather are assemblednon-stochastically. See, e.g., U.S. Pat. Nos. 6,773,900; 6,740,506;6,713,282; 6,635,449; 6,605,449; 6,537,776. In one aspect, SLRcomprises: (a) providing a template polynucleotide, wherein the templatepolynucleotide comprises sequence encoding a homologous gene; (b)providing a plurality of building block polynucleotides, wherein thebuilding block polynucleotides are designed to cross-over reassemblewith the template polynucleotide at a predetermined sequence, and abuilding block polynucleotide comprises a sequence that is a variant ofthe homologous gene and a sequence homologous to the templatepolynucleotide flanking the variant sequence; (c) combining a buildingblock polynucleotide with a template polynucleotide such that thebuilding block polynucleotide cross-over reassembles with the templatepolynucleotide to generate polynucleotides comprising homologous genesequence variations.

SLR does not depend on the presence of high levels of homology betweenpolynucleotides to be rearranged. Thus, this method can be used tonon-stochastically generate libraries (or sets) of progeny moleculescomprised of over 10¹⁰⁰ different chimeras. SLR can be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras. Thus,aspects of the present invention include non-stochastic methods ofproducing a set of finalized chimeric nucleic acid molecule shaving anoverall assembly order that is chosen by design. This method includesthe steps of generating by design a plurality of specific nucleic acidbuilding blocks having serviceable mutually compatible ligatable ends,and assembling these nucleic acid building blocks, such that a designedoverall assembly order is achieved.

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, the overall assembly order in which thenucleic acid building blocks can be coupled is specified by the designof the ligatable ends. If more than one assembly step is to be used,then the overall assembly order in which the nucleic acid buildingblocks can be coupled is also specified by the sequential order of theassembly step(s). In one aspect, the annealed building pieces aretreated with an enzyme, such as a ligase (e.g. T4 DNA ligase), toachieve covalent bonding of the building pieces.

In one aspect, the design of the oligonucleotide building blocks isobtained by analyzing a set of progenitor nucleic acid sequencetemplates that serve as a basis for producing a progeny set of finalizedchimeric polynucleotides. These parental oligonucleotide templates thusserve as a source of sequence information that aids in the design of thenucleic acid building blocks that are to be mutagenized, e.g.,chimerized or shuffled. In one aspect of this method, the sequences of aplurality of parental nucleic acid templates are aligned in order toselect one or more demarcation points. The demarcation points can belocated at an area of homology, and are comprised of one or morenucleotides. These demarcation points are preferably shared by at leasttwo of the progenitor templates. The demarcation points can thereby beused to delineate the boundaries of oligonucleotide building blocks tobe generated in order to rearrange the parental polynucleotides. Thedemarcation points identified and selected in the progenitor moleculesserve as potential chimerization points in the assembly of the finalchimeric progeny molecules. A demarcation point can be an area ofhomology (comprised of at least one homologous nucleotide base) sharedby at least two parental polynucleotide sequences. Alternatively, ademarcation point can be an area of homology that is shared by at leasthalf of the parental polynucleotide sequences, or, it can be an area ofhomology that is shared by at least two thirds of the parentalpolynucleotide sequences. Even more preferably a serviceable demarcationpoints is an area of homology that is shared by at least three fourthsof the parental polynucleotide sequences, or, it can be shared by atalmost all of the parental polynucleotide sequences. In one aspect, ademarcation point is an area of homology that is shared by all of theparental polynucleotide sequences.

In one aspect, a ligation reassembly process is performed exhaustivelyin order to generate an exhaustive library of progeny chimericpolynucleotides. In other words, all possible ordered combinations ofthe nucleic acid building blocks are represented in the set of finalizedchimeric nucleic acid molecules. At the same time, in another aspect,the assembly order (i.e. the order of assembly of each building block inthe 5′ to 3 sequence of each finalized chimeric nucleic acid) in eachcombination is by design (or non-stochastic) as described above. Becauseof the non-stochastic nature of this invention, the possibility ofunwanted side products is greatly reduced.

In another aspect, the ligation reassembly method is performedsystematically. For example, the method is performed in order togenerate a systematically compartmentalized library of progenymolecules, with compartments that can be screened systematically, e.g.one by one. In other words this invention provides that, through theselective and judicious use of specific nucleic acid building blocks,coupled with the selective and judicious use of sequentially steppedassembly reactions, a design can be achieved where specific sets ofprogeny products are made in each of several reaction vessels. Thisallows a systematic examination and screening procedure to be performed.Thus, these methods allow a potentially very large number of progenymolecules to be examined systematically in smaller groups. Because ofits ability to perform chimerizations in a manner that is highlyflexible yet exhaustive and systematic as well, particularly when thereis a low level of homology among the progenitor molecules, these methodsprovide for the generation of a library (or set) comprised of a largenumber of progeny molecules. Because of the non-stochastic nature of theinstant ligation reassembly invention, the progeny molecules generatedpreferably comprise a library of finalized chimeric nucleic acidmolecules having an overall assembly order that is chosen by design. Thesaturation mutagenesis and optimized directed evolution methods also canbe used to generate different progeny molecular species. It isappreciated that the invention provides freedom of choice and controlregarding the selection of demarcation points, the size and number ofthe nucleic acid building blocks, and the size and design of thecouplings. It is appreciated, furthermore, that the requirement forintermolecular homology is highly relaxed for the operability of thisinvention. In fact, demarcation points can even be chosen in areas oflittle or no intermolecular homology. For example, because of codonwobble, i.e. the degeneracy of codons, nucleotide substitutions can beintroduced into nucleic acid building blocks without altering the aminoacid originally encoded in the corresponding progenitor template.Alternatively, a codon can be altered such that the coding for anoriginally amino acid is altered. This invention provides that suchsubstitutions can be introduced into the nucleic acid building block inorder to increase the incidence of intermolecular homologous demarcationpoints and thus to allow an increased number of couplings to be achievedamong the building blocks, which in turn allows a greater number ofprogeny chimeric molecules to be generated.

Synthetic Gene Reassembly

In one aspect, the present invention provides a non-stochastic methodtermed synthetic gene reassembly (e.g., GeneReassembly, see, e.g., U.S.Pat. No. 6,537,776), which differs from stochastic shuffling in that thenucleic acid building blocks are not shuffled or concatenated orchimerized randomly, but rather are assembled non-stochastically.

The synthetic gene reassembly method does not depend on the presence ofa high level of homology between polynucleotides to be shuffled. Theinvention can be used to non-stochastically generate libraries (or sets)of progeny molecules comprised of over 10¹⁰⁰ different chimeras.Conceivably, synthetic gene reassembly can even be used to generatelibraries comprised of over 10¹⁰⁰⁰ different progeny chimeras.

Thus, in one aspect, the invention provides a non-stochastic method ofproducing a set of finalized chimeric nucleic acid molecules having anoverall assembly order that is chosen by design, which method iscomprised of the steps of generating by design a plurality of specificnucleic acid building blocks having serviceable mutually compatibleligatable ends and assembling these nucleic acid building blocks, suchthat a designed overall assembly order is achieved.

In one aspect, synthetic gene reassembly comprises a method of: 1)preparing a progeny generation of molecule(s) (including a moleculecomprising a polynucleotide sequence, e.g., a molecule comprising apolypeptide coding sequence), that is mutagenized to achieve at leastone point mutation, addition, deletion, &/or chimerization, from one ormore ancestral or parental generation template(s); 2) screening theprogeny generation molecule(s), e.g., using a high throughput method,for at least one property of interest (such as an improvement in anenzyme activity); 3) in one aspect obtaining &/or cataloguing structural&/or and functional information regarding the parental &/or progenygeneration molecules; and 4) in one aspect repeating any of steps 1) to3). In one aspect, there is generated (e.g., from a parentpolynucleotide template), in what is termed “codon site-saturationmutagenesis,” a progeny generation of polynucleotides, each having atleast one set of up to three contiguous point mutations (i.e. differentbases comprising a new codon), such that every codon (or every family ofdegenerate codons encoding the same amino acid) is represented at eachcodon position. Corresponding to, and encoded by, this progenygeneration of polynucleotides, there is also generated a set of progenypolypeptides, each having at least one single amino acid point mutation.In a one aspect, there is generated, in what is termed “amino acidsite-saturation mutagenesis”, one such mutant polypeptide for each ofthe 19 naturally encoded polypeptide-forming alpha-amino acidsubstitutions at each and every amino acid position along thepolypeptide. This yields, for each and every amino acid position alongthe parental polypeptide, a total of 20 distinct progeny polypeptidesincluding the original amino acid, or potentially more than 21 distinctprogeny polypeptides if additional amino acids are used either insteadof or in addition to the 20 naturally encoded amino acids

Thus, in another aspect, this approach is also serviceable forgenerating mutants containing, in addition to &/or in combination withthe 20 naturally encoded polypeptide-forming alpha-amino acids, otherrare &/or not naturally-encoded amino acids and amino acid derivatives.In yet another aspect, this approach is also serviceable for generatingmutants by the use of, in addition to &/or in combination with naturalor unaltered codon recognition systems of suitable hosts, altered,mutagenized, &/or designer codon recognition systems (such as in a hostcell with one or more altered tRNA molecules.

In yet another aspect, this invention relates to recombination and morespecifically to a method for preparing polynucleotides encoding apolypeptide by a method of in vivo re-assortment of polynucleotidesequences containing regions of partial homology, assembling thepolynucleotides to form at least one polynucleotide and screening thepolynucleotides for the production of polypeptide(s) having a usefulproperty.

In yet another aspect, this invention is serviceable for analyzing andcataloguing, with respect to any molecular property (e.g. an enzymaticactivity) or combination of properties allowed by current technology,the effects of any mutational change achieved (including particularlysaturation mutagenesis). Thus, a comprehensive method is provided fordetermining the effect of changing each amino acid in a parentalpolypeptide into each of at least 19 possible substitutions. This allowseach amino acid in a parental polypeptide to be characterized andcatalogued according to its spectrum of potential effects on ameasurable property of the polypeptide.

In one aspect, an intron may be introduced into a chimeric progenymolecule by way of a nucleic acid building block. Introns often haveconsensus sequences at both termini in order to render them operational.In addition to enabling gene splicing, introns may serve an additionalpurpose by providing sites of homology to other nucleic acids to enablehomologous recombination. For this purpose, and potentially others, itmay be sometimes desirable to generate a large nucleic acid buildingblock for introducing an intron. If the size is overly large easilygenerating by direct chemical synthesis of two single stranded oligos,such a specialized nucleic acid building block may also be generated bydirect chemical synthesis of more than two single stranded oligos or byusing a polymerase-based amplification reaction

The mutually compatible ligatable ends of the nucleic acid buildingblocks to be assembled are considered to be “serviceable” for this typeof ordered assembly if they enable the building blocks to be coupled inpredetermined orders. Thus, in one aspect, the overall assembly order inwhich the nucleic acid building blocks can be coupled is specified bythe design of the ligatable ends and, if more than one assembly step isto be used, then the overall assembly order in which the nucleic acidbuilding blocks can be coupled is also specified by the sequential orderof the assembly step(s). In a one aspect of the invention, the annealedbuilding pieces are treated with an enzyme, such as a ligase (e.g., T4DNA ligase) to achieve covalent bonding of the building pieces.

Coupling can occur in a manner that does not make use of everynucleotide in a participating overhang. The coupling is particularlylively to survive (e.g. in a transformed host) if the couplingreinforced by treatment with a ligase enzyme to form what may bereferred to as a “gap ligation” or a “gapped ligation”. This type ofcoupling can contribute to generation of unwanted background product(s),but it can also be used advantageously increase the diversity of theprogeny library generated by the designed ligation reassembly. Certainoverhangs are able to undergo self-coupling to form a palindromiccoupling. A coupling is strengthened substantially if it is reinforcedby treatment with a ligase enzyme. Lack of 5′ phosphates on theseoverhangs can be used advantageously to prevent this type of palindromicself-ligation. Accordingly, this invention provides that nucleic acidbuilding blocks can be chemically made (or ordered) that lack a 5′phosphate group. Alternatively, they can be removed, e.g. by treatmentwith a phosphatase enzyme, such as a calf intestinal alkalinephosphatase (CIAP), in order to prevent palindromic self-ligations inligation reassembly processes.

In a another aspect, the design of nucleic acid building blocks isobtained upon analysis of the sequences of a set of progenitor nucleicacid templates that serve as a basis for producing a progeny set offinalized chimeric nucleic acid molecules. These progenitor nucleic acidtemplates thus serve as a source of sequence information that aids inthe design of the nucleic acid building blocks that are to bemutagenized, i.e. chimerized or shuffled.

In one exemplification, the invention provides for the chimerization ofa family of related genes and their encoded family of related products.In a particular exemplification, the encoded products are enzymes. Theisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the present invention can be mutagenizedin accordance with the methods described herein.

Thus according to one aspect of the invention, the sequences of aplurality of progenitor nucleic acid templates (e.g., polynucleotides ofThe invention) are aligned in order to select one or more demarcationpoints, which demarcation points can be located at an area of homology.The demarcation points can be used to delineate the boundaries ofnucleic acid building blocks to be generated. Thus, the demarcationpoints identified and selected in the progenitor molecules serve aspotential chimerization points in the assembly of the progeny molecules.

Typically a serviceable demarcation point is an area of homology(comprised of at least one homologous nucleotide base) shared by atleast two progenitor templates, but the demarcation point can be an areaof homology that is shared by at least half of the progenitor templates,at least two thirds of the progenitor templates, at least three fourthsof the progenitor templates and preferably at almost all of theprogenitor templates. Even more preferably still a serviceabledemarcation point is an area of homology that is shared by all of theprogenitor templates.

In a one aspect, the gene reassembly process is performed exhaustivelyin order to generate an exhaustive library. In other words, all possibleordered combinations of the nucleic acid building blocks are representedin the set of finalized chimeric nucleic acid molecules. At the sametime, the assembly order (i.e. the order of assembly of each buildingblock in the 5′ to 3 sequence of each finalized chimeric nucleic acid)in each combination is by design (or non-stochastic). Because of thenon-stochastic nature of the method, the possibility of unwanted sideproducts is greatly reduced.

In another aspect, the method provides that the gene reassembly processis performed systematically, for example to generate a systematicallycompartmentalized library, with compartments that can be screenedsystematically, e.g., one by one. In other words the invention providesthat, through the selective and judicious use of specific nucleic acidbuilding blocks, coupled with the selective and judicious use ofsequentially stepped assembly reactions, an experimental design can beachieved where specific sets of progeny products are made in each ofseveral reaction vessels. This allows a systematic examination andscreening procedure to be performed. Thus, it allows a potentially verylarge number of progeny molecules to be examined systematically insmaller groups.

Because of its ability to perform chimerizations in a manner that ishighly flexible yet exhaustive and systematic as well, particularly whenthere is a low level of homology among the progenitor molecules, theinstant invention provides for the generation of a library (or set)comprised of a large number of progeny molecules. Because of thenon-stochastic nature of the instant gene reassembly invention, theprogeny molecules generated preferably comprise a library of finalizedchimeric nucleic acid molecules having an overall assembly order that ischosen by design. In a particularly aspect, such a generated library iscomprised of greater than 10³ to greater than 10¹⁰⁰⁰ different progenymolecular species.

In one aspect, a set of finalized chimeric nucleic acid molecules,produced as described is comprised of a polynucleotide encoding apolypeptide. According to one aspect, this polynucleotide is a gene,which may be a man-made gene. According to another aspect, thispolynucleotide is a gene pathway, which may be a man-made gene pathway.The invention provides that one or more man-made genes generated by theinvention may be incorporated into a man-made gene pathway, such aspathway operable in a eukaryotic organism (including a plant).

In another exemplification, the synthetic nature of the step in whichthe building blocks are generated allows the design and introduction ofnucleotides (e.g., one or more nucleotides, which may be, for example,codons or introns or regulatory sequences) that can later be in oneaspect removed in an in vitro process (e.g., by mutagenesis) or in an invivo process (e.g., by utilizing the gene splicing ability of a hostorganism). It is appreciated that in many instances the introduction ofthese nucleotides may also be desirable for many other reasons inaddition to the potential benefit of creating a serviceable demarcationpoint.

Thus, according to another aspect, the invention provides that a nucleicacid building block can be used to introduce an intron. Thus, theinvention provides that functional introns may be introduced into aman-made gene of the invention. The invention also provides thatfunctional introns may be introduced into a man-made gene pathway of theinvention. Accordingly, the invention provides for the generation of achimeric polynucleotide that is a man-made gene containing one (or more)artificially introduced intron(s).

Accordingly, the invention also provides for the generation of achimeric polynucleotide that is a man-made gene pathway containing one(or more) artificially introduced intron(s). Preferably, theartificially introduced intron(s) are functional in one or more hostcells for gene splicing much in the way that naturally-occurring intronsserve functionally in gene splicing. The invention provides a process ofproducing man-made intron-containing polynucleotides to be introducedinto host organisms for recombination and/or splicing.

A man-made gene produced using the invention can also serve as asubstrate for recombination with another nucleic acid. Likewise, aman-made gene pathway produced using the invention can also serve as asubstrate for recombination with another nucleic acid. In a one aspect,the recombination is facilitated by, or occurs at, areas of homologybetween the man-made, intron-containing gene and a nucleic acid, whichserves as a recombination partner. In one aspect, the recombinationpartner may also be a nucleic acid generated by the invention, includinga man-made gene or a man-made gene pathway. Recombination may befacilitated by or may occur at areas of homology that exist at the one(or more) artificially introduced intron(s) in the man-made gene.

The synthetic gene reassembly method of the invention utilizes aplurality of nucleic acid building blocks, each of which preferably hastwo ligatable ends. The two ligatable ends on each nucleic acid buildingblock may be two blunt ends (i.e. each having an overhang of zeronucleotides), or preferably one blunt end and one overhang, or morepreferably still two overhangs.

A useful overhang for this purpose may be a 3′ overhang or a 5′overhang. Thus, a nucleic acid building block may have a 3′ overhang oralternatively a 5′ overhang or alternatively two 3′ overhangs oralternatively two 5′ overhangs. The overall order in which the nucleicacid building blocks are assembled to form a finalized chimeric nucleicacid molecule is determined by purposeful experimental design and is notrandom.

In one aspect, a nucleic acid building block is generated by chemicalsynthesis of two single-stranded nucleic acids (also referred to assingle-stranded oligos) and contacting them so as to allow them toanneal to form a double-stranded nucleic acid building block.

A double-stranded nucleic acid building block can be of variable size.The sizes of these building blocks can be small or large. Exemplarysizes for building block range from 1 base pair (not including anyoverhangs) to 100,000 base pairs (not including any overhangs). Otherexemplary size ranges are also provided, which have lower limits of from1 bp to 10,000 bp (including every integer value in between) and upperlimits of from 2 bp to 100,000 bp (including every integer value inbetween).

Many methods exist by which a double-stranded nucleic acid buildingblock can be generated that is serviceable for the invention; and theseare known in the art and can be readily performed by the skilledartisan.

According to one aspect, a double-stranded nucleic acid building blockis generated by first generating two single stranded nucleic acids andallowing them to anneal to form a double-stranded nucleic acid buildingblock. The two strands of a double-stranded nucleic acid building blockmay be complementary at every nucleotide apart from any that form anoverhang; thus containing no mismatches, apart from any overhang(s).According to another aspect, the two strands of a double-strandednucleic acid building block are complementary at fewer than everynucleotide apart from any that form an overhang. Thus, according to thisaspect, a double-stranded nucleic acid building block can be used tointroduce codon degeneracy. The codon degeneracy can be introduced usingthe site-saturation mutagenesis described herein, using one or moreN,N,G/T cassettes or alternatively using one or more N,N,N cassettes.

The in vivo recombination method of the invention can be performedblindly on a pool of unknown hybrids or alleles of a specificpolynucleotide or sequence. However, it is not necessary to know theactual DNA or RNA sequence of the specific polynucleotide.

The approach of using recombination within a mixed population of genescan be useful for the generation of any useful proteins, for example,interleukin I, antibodies, tPA and growth hormone. This approach may beused to generate proteins having altered specificity or activity. Theapproach may also be useful for the generation of hybrid nucleic acidsequences, for example, promoter regions, introns, exons, enhancersequences, 31 untranslated regions or 51 untranslated regions of genes.Thus this approach may be used to generate genes having increased ratesof expression. This approach may also be useful in the study ofrepetitive DNA sequences. Finally, this approach may be useful to mutateribozymes or aptamers.

In one aspect the invention described herein is directed to the use ofrepeated cycles of reductive reassortment, recombination and selectionwhich allow for the directed molecular evolution of highly complexlinear sequences, such as DNA, RNA or proteins thorough recombination.

Optimized Directed Evolution System

The invention provides a non-stochastic gene modification system termed“optimized directed evolution system” to generate polypeptides, e.g.,isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases, or antibodies of the invention, with newor altered properties. Optimized directed evolution is directed to theuse of repeated cycles of reductive reassortment, recombination andselection that allow for the directed molecular evolution of nucleicacids through recombination. Optimized directed evolution allowsgeneration of a large population of evolved chimeric sequences, whereinthe generated population is significantly enriched for sequences thathave a predetermined number of crossover events.

A crossover event is a point in a chimeric sequence where a shift insequence occurs from one parental variant to another parental variant.Such a point is normally at the juncture of where oligonucleotides fromtwo parents are ligated together to form a single sequence. This methodallows calculation of the correct concentrations of oligonucleotidesequences so that the final chimeric population of sequences is enrichedfor the chosen number of crossover events. This provides more controlover choosing chimeric variants having a predetermined number ofcrossover events.

In addition, this method provides a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. Previously, if one generated, for example, 10¹³ chimericmolecules during a reaction, it would be extremely difficult to testsuch a high number of chimeric variants for a particular activity.Moreover, a significant portion of the progeny population would have avery high number of crossover events which resulted in proteins thatwere less likely to have increased levels of a particular activity. Byusing these methods, the population of chimerics molecules can beenriched for those variants that have a particular number of crossoverevents. Thus, although one can still generate 10¹³ chimeric moleculesduring a reaction, each of the molecules chosen for further analysismost likely has, for example, only three crossover events. Because theresulting progeny population can be skewed to have a predeterminednumber of crossover events, the boundaries on the functional varietybetween the chimeric molecules is reduced. This provides a moremanageable number of variables when calculating which oligonucleotidefrom the original parental polynucleotides might be responsible foraffecting a particular trait.

One method for creating a chimeric progeny polynucleotide sequence is tocreate oligonucleotides corresponding to fragments or portions of eachparental sequence. Each oligonucleotide preferably includes a uniqueregion of overlap so that mixing the oligonucleotides together resultsin a new variant that has each oligonucleotide fragment assembled in thecorrect order. Additional information can also be found, e.g., in U.S.Ser. No. 09/332,835; U.S. Pat. No. 6,361,974.

The number of oligonucleotides generated for each parental variant bearsa relationship to the total number of resulting crossovers in thechimeric molecule that is ultimately created. For example, threeparental nucleotide sequence variants might be provided to undergo aligation reaction in order to find a chimeric variant having, forexample, greater activity at high temperature. As one example, a set of50 oligonucleotide sequences can be generated corresponding to eachportions of each parental variant. Accordingly, during the ligationreassembly process there could be up to 50 crossover events within eachof the chimeric sequences. The probability that each of the generatedchimeric polynucleotides will contain oligonucleotides from eachparental variant in alternating order is very low. If eacholigonucleotide fragment is present in the ligation reaction in the samemolar quantity it is likely that in some positions oligonucleotides fromthe same parental polynucleotide will ligate next to one another andthus not result in a crossover event. If the concentration of eacholigonucleotide from each parent is kept constant during any ligationstep in this example, there is a ⅓ chance (assuming 3 parents) that anoligonucleotide from the same parental variant will ligate within thechimeric sequence and produce no crossover.

Accordingly, a probability density function (PDF) can be determined topredict the population of crossover events that are likely to occurduring each step in a ligation reaction given a set number of parentalvariants, a number of oligonucleotides corresponding to each variant,and the concentrations of each variant during each step in the ligationreaction. The statistics and mathematics behind determining the PDF isdescribed below. By utilizing these methods, one can calculate such aprobability density function, and thus enrich the chimeric progenypopulation for a predetermined number of crossover events resulting froma particular ligation reaction. Moreover, a target number of crossoverevents can be predetermined, and the system then programmed to calculatethe starting quantities of each parental oligonucleotide during eachstep in the ligation reaction to result in a probability densityfunction that centers on the predetermined number of crossover events.These methods are directed to the use of repeated cycles of reductivereassortment, recombination and selection that allow for the directedmolecular evolution of a nucleic acid encoding a polypeptide throughrecombination. This system allows generation of a large population ofevolved chimeric sequences, wherein the generated population issignificantly enriched for sequences that have a predetermined number ofcrossover events. A crossover event is a point in a chimeric sequencewhere a shift in sequence occurs from one parental variant to anotherparental variant. Such a point is normally at the juncture of whereoligonucleotides from two parents are ligated together to form a singlesequence. The method allows calculation of the correct concentrations ofoligonucleotide sequences so that the final chimeric population ofsequences is enriched for the chosen number of crossover events. Thisprovides more control over choosing chimeric variants having apredetermined number of crossover events.

In addition, these methods provide a convenient means for exploring atremendous amount of the possible protein variant space in comparison toother systems. By using the methods described herein, the population ofchimerics molecules can be enriched for those variants that have aparticular number of crossover events. Thus, although one can stillgenerate 10¹³ chimeric molecules during a reaction, each of themolecules chosen for further analysis most likely has, for example, onlythree crossover events. Because the resulting progeny population can beskewed to have a predetermined number of crossover events, theboundaries on the functional variety between the chimeric molecules isreduced. This provides a more manageable number of variables whencalculating which oligonucleotide from the original parentalpolynucleotides might be responsible for affecting a particular trait.

In one aspect, the method creates a chimeric progeny polynucleotidesequence by creating oligonucleotides corresponding to fragments orportions of each parental sequence. Each oligonucleotide preferablyincludes a unique region of overlap so that mixing the oligonucleotidestogether results in a new variant that has each oligonucleotide fragmentassembled in the correct order. See also U.S. Ser. No. 09/332,835.

Determining Crossover Events

Aspects of the invention include a system and software that receive adesired crossover probability density function (PDF), the number ofparent genes to be reassembled, and the number of fragments in thereassembly as inputs. The output of this program is a “fragment PDF”that can be used to determine a recipe for producing reassembled genes,and the estimated crossover PDF of those genes. The processing describedherein is preferably performed in MATLAB™ (The Mathworks, Natick, Mass.)a programming language and development environment for technicalcomputing.

Iterative Processes

In practicing the invention, these processes can be iterativelyrepeated. For example, a nucleic acid (or, the nucleic acid) responsiblefor an altered or new isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase phenotype is identified,re-isolated, again modified, re-tested for activity. This process can beiteratively repeated until a desired phenotype is engineered. Forexample, an entire biochemical anabolic or catabolic pathway can beengineered into a cell, including, e.g., isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase activity.

Similarly, if it is determined that a particular oligonucleotide has noaffect at all on the desired trait (e.g., a new isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasephenotype), it can be removed as a variable by synthesizing largerparental oligonucleotides that include the sequence to be removed. Sinceincorporating the sequence within a larger sequence prevents anycrossover events, there will no longer be any variation of this sequencein the progeny polynucleotides. This iterative practice of determiningwhich oligonucleotides are most related to the desired trait, and whichare unrelated, allows more efficient exploration all of the possibleprotein variants that might be provide a particular trait or activity.

In Vivo Shuffling

In vivo shuffling of molecules is use in methods of the invention thatprovide variants of polypeptides of the invention, e.g., antibodies,isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases, and the like. In vivo shuffling can beperformed utilizing the natural property of cells to recombinemultimers. While recombination in vivo has provided the major naturalroute to molecular diversity, genetic recombination remains a relativelycomplex process that involves 1) the recognition of homologies; 2)strand cleavage, strand invasion, and metabolic steps leading to theproduction of recombinant chiasma; and finally 3) the resolution ofchiasma into discrete recombined molecules. The formation of the chiasmarequires the recognition of homologous sequences.

In another aspect, the invention includes a method for producing ahybrid polynucleotide from at least a first polynucleotide and a secondpolynucleotide. The invention can be used to produce a hybridpolynucleotide by introducing at least a first polynucleotide and asecond polynucleotide which share at least one region of partialsequence homology into a suitable host cell. The regions of partialsequence homology promote processes which result in sequencereorganization producing a hybrid polynucleotide. The term “hybridpolynucleotide”, as used herein, is any nucleotide sequence whichresults from the method of the present invention and contains sequencefrom at least two original polynucleotide sequences. Such hybridpolynucleotides can result from intermolecular recombination eventswhich promote sequence integration between DNA molecules. In addition,such hybrid polynucleotides can result from intramolecular reductivereassortment processes which utilize repeated sequences to alter anucleotide sequence within a DNA molecule.

In vivo reassortment is focused on “inter-molecular” processescollectively referred to as “recombination” which in bacteria, isgenerally viewed as a “RecA-dependent” phenomenon. The invention canrely on recombination processes of a host cell to recombine andre-assort sequences, or the cells' ability to mediate reductiveprocesses to decrease the complexity of quasi-repeated sequences in thecell by deletion. This process of “reductive reassortment” occurs by an“intra-molecular”, RecA-independent process.

Therefore, in another aspect of the invention, novel polynucleotides canbe generated by the process of reductive reassortment. The methodinvolves the generation of constructs containing consecutive sequences(original encoding sequences), their insertion into an appropriatevector and their subsequent introduction into an appropriate host cell.The reassortment of the individual molecular identities occurs bycombinatorial processes between the consecutive sequences in theconstruct possessing regions of homology, or between quasi-repeatedunits. The reassortment process recombines and/or reduces the complexityand extent of the repeated sequences and results in the production ofnovel molecular species. Various treatments may be applied to enhancethe rate of reassortment. These could include treatment withultra-violet light, or DNA damaging chemicals and/or the use of hostcell lines displaying enhanced levels of “genetic instability”. Thus thereassortment process may involve homologous recombination or the naturalproperty of quasi-repeated sequences to direct their own evolution.

Repeated or “quasi-repeated” sequences play a role in geneticinstability. In the present invention, “quasi-repeats” are repeats thatare not restricted to their original unit structure. Quasi-repeatedunits can be presented as an array of sequences in a construct;consecutive units of similar sequences. Once ligated, the junctionsbetween the consecutive sequences become essentially invisible and thequasi-repetitive nature of the resulting construct is now continuous atthe molecular level. The deletion process the cell performs to reducethe complexity of the resulting construct operates between thequasi-repeated sequences. The quasi-repeated units provide a practicallylimitless repertoire of templates upon which slippage events can occur.The constructs containing the quasi-repeats thus effectively providesufficient molecular elasticity that deletion (and potentiallyinsertion) events can occur virtually anywhere within thequasi-repetitive units.

When the quasi-repeated sequences are all ligated in the sameorientation, for instance head to tail or vice versa, the cell cannotdistinguish individual units. Consequently, the reductive process canoccur throughout the sequences. In contrast, when for example, the unitsare presented head to head, rather than head to tail, the inversiondelineates the endpoints of the adjacent unit so that deletion formationwill favor the loss of discrete units. Thus, it is preferable with thepresent method that the sequences are in the same orientation. Randomorientation of quasi-repeated sequences will result in the loss ofreassortment efficiency, while consistent orientation of the sequenceswill offer the highest efficiency. However, while having fewer of thecontiguous sequences in the same orientation decreases the efficiency,it may still provide sufficient elasticity for the effective recovery ofnovel molecules. Constructs can be made with the quasi-repeatedsequences in the same orientation to allow higher efficiency.

Sequences can be assembled in a head to tail orientation using any of avariety of methods, including the following:

-   -   a) Primers that include a poly-A head and poly-T tail which when        made single-stranded would provide orientation can be utilized.        This is accomplished by having the first few bases of the        primers made from RNA and hence easily removed RNaseH.    -   b) Primers that include unique restriction cleavage sites can be        utilized. Multiple sites, a battery of unique sequences and        repeated synthesis and ligation steps would be required.    -   c) The inner few bases of the primer could be thiolated and an        exonuclease used to produce properly tailed molecules.

The recovery of the re-assorted sequences relies on the identificationof cloning vectors with a reduced repetitive index (RI). The re-assortedencoding sequences can then be recovered by amplification. The productsare re-cloned and expressed. The recovery of cloning vectors withreduced RI can be affected by:

-   1) The use of vectors only stably maintained when the construct is    reduced in complexity.-   2) The physical recovery of shortened vectors by physical    procedures. In this case, the cloning vector would be recovered    using standard plasmid isolation procedures and size fractionated on    either an agarose gel, or column with a low molecular weight cut off    utilizing standard procedures.-   3) The recovery of vectors containing interrupted genes which can be    selected when insert size decreases.-   4) The use of direct selection techniques with an expression vector    and the appropriate selection.

Encoding sequences (for example, genes) from related organisms maydemonstrate a high degree of homology and encode quite diverse proteinproducts. These types of sequences are particularly useful in thepresent invention as quasi-repeats. However, while the examplesillustrated below demonstrate the reassortment of nearly identicaloriginal encoding sequences (quasi-repeats), this process is not limitedto such nearly identical repeats.

The following example demonstrates a method of the invention. Encodingnucleic acid sequences (quasi-repeats) derived from three (3) uniquespecies are described. Each sequence encodes a protein with a distinctset of properties. Each of the sequences differs by a single or a fewbase pairs at a unique position in the sequence. The quasi-repeatedsequences are separately or collectively amplified and ligated intorandom assemblies such that all possible permutations and combinationsare available in the population of ligated molecules. The number ofquasi-repeat units can be controlled by the assembly conditions. Theaverage number of quasi-repeated units in a construct is defined as therepetitive index (RI).

Once formed, the constructs may, or may not be size fractionated on anagarose gel according to published protocols, inserted into a cloningvector and transfected into an appropriate host cell. The cells are thenpropagated and “reductive reassortment” is effected. The rate of thereductive reassortment process may be stimulated by the introduction ofDNA damage if desired. Whether the reduction in RI is mediated bydeletion formation between repeated sequences by an “intra-molecular”mechanism, or mediated by recombination-like events through“inter-molecular” mechanisms is immaterial. The end result is areassortment of the molecules into all possible combinations.

In one aspect (optionally), the method comprises the additional step ofscreening the library members of the shuffled pool to identifyindividual shuffled library members having the ability to bind orotherwise interact, or catalyze a particular reaction (e.g., such ascatalytic domain of an enzyme) with a predetermined macromolecule, suchas for example a proteinaceous receptor, an oligosaccharide, virion, orother predetermined compound or structure.

The polypeptides that are identified from such libraries can be used fortherapeutic, diagnostic, research and related purposes (e.g., catalysts,solutes for increasing osmolarity of an aqueous solution and the like)and/or can be subjected to one or more additional cycles of shufflingand/or selection.

In another aspect, it is envisioned that prior to or duringrecombination or reassortment, polynucleotides generated by the methodof the invention can be subjected to agents or processes which promotethe introduction of mutations into the original polynucleotides. Theintroduction of such mutations would increase the diversity of resultinghybrid polynucleotides and polypeptides encoded therefrom. The agents orprocesses which promote mutagenesis can include, but are not limited to:(+)-CC-1065, or a synthetic analog such as (+)-CC-1065-(N3-Adenine (SeeSun and Hurley, (1992); an N-acetylated or deacetylated4′-fluoro-4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See™, for example, van de Poll et al. (1992)); or a N-acetylated ordeacetylated 4-aminobiphenyl adduct capable of inhibiting DNA synthesis(See also, van de Poll et al. (1992), pp. 751-758); trivalent chromium,a trivalent chromium salt, a polycyclic aromatic hydrocarbon (PAH) DNAadduct capable of inhibiting DNA replication, such as7-bromomethyl-benz[a]anthracene (“BMA”),tris(2,3-dibromopropyl)phosphate (“Tris-BP”),1,2-dibromo-3-chloropropane (“DBCP”), 2-bromoacrolein (2BA),benzo[a]pyrene-7,8-dihydrodiol-9-10-epoxide (“BPDE”), a platinum(II)halogen salt, N-hydroxy-2-amino-3-methylimidazo[4,5-f]-quinoline(“N-hydroxy-IQ”) andN-hydroxy-2-amino-1-methyl-6-phenylimidazo[4,5-f]-pyridine(“N-hydroxy-PhIP”). Exemplary means for slowing or halting PCRamplification consist of UV light (+)-CC-1065 and(+)-CC-1065-(N3-Adenine). Particularly encompassed means are DNA adductsor polynucleotides comprising the DNA adducts from the polynucleotidesor polynucleotides pool, which can be released or removed by a processincluding heating the solution comprising the polynucleotides prior tofurther processing.

In another aspect the invention is directed to a method of producingrecombinant proteins having biological activity by treating a samplecomprising double-stranded template polynucleotides encoding a wild-typeprotein under conditions according to the invention which provide forthe production of hybrid or re-assorted polynucleotides.

Producing Sequence Variants

The invention also provides additional methods for making sequencevariants of the nucleic acid (e.g., isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase) sequences ofthe invention. The invention also provides additional methods forisolating isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases using the nucleic acids andpolypeptides of the invention. In one aspect, the invention provides forvariants of an isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase coding sequence (e.g., a gene, cDNAor message) of the invention, which can be altered by any means,including, e.g., random or stochastic methods, or, non-stochastic, or“directed evolution,” methods, as described above.

The isolated variants may be naturally occurring. Variant can also becreated in vitro. Variants may be created using genetic engineeringtechniques such as site directed mutagenesis, random chemicalmutagenesis, Exonuclease III deletion procedures, and standard cloningtechniques. Alternatively, such variants, fragments, analogs, orderivatives may be created using chemical synthesis or modificationprocedures. Other methods of making variants are also familiar to thoseskilled in the art. These include procedures in which nucleic acidsequences obtained from natural isolates are modified to generate newnucleic acids which encode polypeptides having characteristics whichenhance their value in industrial, medical, laboratory (research),pharmaceutical, food and feed and food and feed supplement processingand other applications and processes. In such procedures, a large numberof variant sequences having one or more nucleotide differences withrespect to the sequence obtained from the natural isolate are generatedand characterized. These nucleotide differences can result in amino acidchanges with respect to the polypeptides encoded by the nucleic acidsfrom the natural isolates.

For example, variants may be created using error prone PCR. In errorprone PCR, PCR is performed under conditions where the copying fidelityof the DNA polymerase is low, such that a high rate of point mutationsis obtained along the entire length of the PCR product. Error prone PCRis described, e.g., in Leung, D. W., et al., Technique, 1:11-15, 1989)and Caldwell, R. C. & Joyce G. F., PCR Methods Applic., 2:28-33, 1992.Briefly, in such procedures, nucleic acids to be mutagenized are mixedwith PCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction may be performed using 20 fmoles of nucleic acid to bemutagenized, 30 pmole of each PCR primer, a reaction buffer comprising50 mM KCl, 10 mM Tris HCl (pH 8.3) and 0.01% gelatin, 7 mM MgCl₂, 0.5 mMMnCl₂, 5 units of Taq polymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP,and 1 mM dTTP. PCR may be performed for 30 cycles of 94° C. for 1 min,45° C. for 1 min, and 72° C. for 1 min. However, it will be appreciatedthat these parameters may be varied as appropriate. The mutagenizednucleic acids are cloned into an appropriate vector and the activitiesof the polypeptides encoded by the mutagenized nucleic acids areevaluated.

Variants may also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described, e.g., in Reidhaar-Olson (1988)Science 241:53-57. Briefly, in such procedures a plurality of doublestranded oligonucleotides bearing one or more mutations to be introducedinto the cloned DNA are synthesized and inserted into the cloned DNA tobe mutagenized. Clones containing the mutagenized DNA are recovered andthe activities of the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, e.g., U.S. Pat. No.5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different but highly related DNA sequence invitro, as a result of random fragmentation of the DNA molecule based onsequence homology, followed by fixation of the crossover by primerextension in a PCR reaction. Sexual PCR mutagenesis is described, e.g.,in Stemmer (1994) Proc. Natl. Acad. Sci. USA 91:10747-10751. Briefly, insuch procedures a plurality of nucleic acids to be recombined aredigested with DNase to generate fragments having an average size of50-200 nucleotides. Fragments of the desired average size are purifiedand resuspended in a PCR mixture. PCR is conducted under conditionswhich facilitate recombination between the nucleic acid fragments. Forexample, PCR may be performed by resuspending the purified fragments ata concentration of 10-30 ng/μl in a solution of 0.2 mM of each dNTP, 2.2mM MgCl₂, 50 mM KCL, 10 mM Tris HCl, pH 9.0, and 0.1% Triton X-100. 2.5units of Taq polymerase per 100:1 of reaction mixture is added and PCRis performed using the following regime: 94° C. for 60 seconds, 94° C.for 30 seconds, 50-55° C. for 30 seconds, 72° C. for 30 seconds (30-45times) and 72° C. for 5 minutes. However, it will be appreciated thatthese parameters may be varied as appropriate. In some aspects,oligonucleotides may be included in the PCR reactions. In other aspects,the Klenow fragment of DNA polymerase I may be used in a first set ofPCR reactions and Taq polymerase may be used in a subsequent set of PCRreactions. Recombinant sequences are isolated and the activities of thepolypeptides they encode are assessed.

Variants may also be created by in vivo mutagenesis. In some aspects,random mutations in a sequence of interest are generated by propagatingthe sequence of interest in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type parent. Propagating the DNA in one of these strainswill eventually generate random mutations within the DNA. Mutatorstrains suitable for use for in vivo mutagenesis are described in PCTPublication No. WO 91/16427, published Oct. 31, 1991, entitled “Methodsfor Phenotype Creation from Multiple Gene Populations”.

Variants may also be generated using cassette mutagenesis. In cassettemutagenesis a small region of a double stranded DNA molecule is replacedwith a synthetic oligonucleotide “cassette” that differs from the nativesequence. The oligonucleotide often contains completely and/or partiallyrandomized native sequence.

Recursive ensemble mutagenesis may also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in Arkin, A. P. and Youvan, D. C., PNAS, USA,89:7811-7815, 1992.

In some aspects, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is described inDelegrave, S. and Youvan, D. C., Biotechnology Research, 11:1548-1552,1993. Random and site-directed mutagenesis are described in Arnold, F.H., Current Opinion in Biotechnology, 4:450-455, 1993.

In some aspects, the variants are created using shuffling procedureswherein portions of a plurality of nucleic acids which encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences which encode chimeric polypeptides as described in U.S. Pat.No. 5,965,408, filed Jul. 9, 1996, entitled, “Method of DNA Reassemblyby Interrupting Synthesis” and U.S. Pat. No. 5,939,250, filed May 22,1996, entitled, “Production of Enzymes Having Desired Activities byMutagenesis.

The variants of the polypeptides of the invention may be variants inwhich one or more of the amino acid residues of the polypeptides of theinvention are substituted with a conserved or non-conserved amino acidresidue (preferably a conserved amino acid residue) and such substitutedamino acid residue may or may not be one encoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the followingreplacements: replacements of an aliphatic amino acid such as Alanine,Valine, Leucine and Isoleucine with another aliphatic amino acid;replacement of a Serine with a Threonine or vice versa; replacement ofan acidic residue such as Aspartic acid and Glutamic acid with anotheracidic residue; replacement of a residue bearing an amide group, such asAsparagine and Glutamine, with another residue bearing an amide group;exchange of a basic residue such as Lysine and Arginine with anotherbasic residue; and replacement of an aromatic residue such asPhenylalanine, Tyrosine with another aromatic residue.

Other variants are those in which one or more of the amino acid residuesof the polypeptides of the invention includes a substituent group.

Still other variants are those in which the polypeptide is associatedwith another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol).

Additional variants are those in which additional amino acids are fusedto the polypeptide, such as a leader sequence, a secretory sequence, aproprotein sequence or a sequence which facilitates purification,enrichment, or stabilization of the polypeptide.

In some aspects, the fragments, derivatives and analogs retain the samebiological function or activity as the polypeptides of the invention andsequences substantially identical thereto. In other aspects, thefragment, derivative, or analog includes a proprotein, such that thefragment, derivative, or analog can be activated by cleavage of theproprotein portion to produce an active polypeptide.

Optimizing Codons to Achieve High Levels of Protein Expression in HostCells

The invention provides methods for modifying an isomerase-, e.g., aracemase-, e.g., an amino acid racemase-, an alanine racemase-, and/oran epimerase-isomerase-, e.g., racemase-, e.g., amino acid racemase-,alanine racemase-, and/or epimerase-encoding nucleic acids to modifycodon usage. In one aspect, the invention provides methods for modifyingcodons in a nucleic acid encoding an isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase to increase ordecrease its expression in a host cell. The invention also providesnucleic acids encoding an isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase modified to increase itsexpression in a host cell, enzymes so modified, and methods of makingthe modified enzymes. The method comprises identifying a “non-preferred”or a “less preferred” codon in an isomerase-, e.g., racemase-, e.g.,amino acid racemase-, alanine racemase-, and/or epimerase-encodingnucleic acid and replacing one or more of these non-preferred or lesspreferred codons with a “preferred codon” encoding the same amino acidas the replaced codon and at least one non-preferred or less preferredcodon in the nucleic acid has been replaced by a preferred codonencoding the same amino acid. A preferred codon is a codonover-represented in coding sequences in genes in the host cell and anon-preferred or less preferred codon is a codon under-represented incoding sequences in genes in the host cell.

Host cells for expressing the nucleic acids, expression cassettes andvectors of the invention include bacteria, yeast, fungi, plant cells,insect cells and mammalian cells. Thus, the invention provides methodsfor optimizing codon usage in all of these cells, codon-altered nucleicacids and polypeptides made by the codon-altered nucleic acids.Exemplary host cells include gram negative bacteria, such as Escherichiacoli and Pseudomonas fluorescens; gram positive bacteria, such asLactobacillus gasseri, Lactococcus lactis, Lactococcus cremoris,Bacillus subtilis. Exemplary host cells also include eukaryoticorganisms, e.g., various yeast, such as Saccharomyces sp., includingSaccharomyces cerevisiae, Schizosaccharomyces pombe, Pichia pastoris,and Kluyveromyces lactis, Hansenula polymorpha, Aspergillus niger, andmammalian cells and cell lines and insect cells and cell lines. Otherexemplary host cells include bacterial cells, such as E. coli,Streptomyces, Bacillus subtilis, Bacillus cereus, Salmonella typhimuriumand various species within the genera Pseudomonas, Streptomyces andStaphylococcus, fungal cells, such as Aspergillus, yeast such as anyspecies of Pichia, Saccharomyces, Schizosaccharomyces, Schwanniomyces,including Pichia pastoris, Saccharomyces cerevisiae, orSchizosaccharomyces pombe, insect cells such as Drosophila S2 andSpodoptera Sf9, animal cells such as CHO, COS or Bowes melanoma andadenoviruses. The selection of an appropriate host is within theabilities of those skilled in the art. Thus, the invention also includesnucleic acids and polypeptides optimized for expression in theseorganisms and species.

For example, the codons of a nucleic acid encoding an isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseisolated from a bacterial cell are modified such that the nucleic acidis optimally expressed in a bacterial cell different from the bacteriafrom which the isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase was derived, a yeast, a fungi, aplant cell, an insect cell or a mammalian cell. Methods for optimizingcodons are well known in the art, see, e.g., U.S. Pat. No. 5,795,737;Baca (2000) Int. J. Parasitol. 30:113-118; Hale (1998) Protein Expr.Purif. 12:185-188; Narum (2001) Infect. Immun. 69:7250-7253. See alsoNarum (2001) Infect. Immun. 69:7250-7253, describing optimizing codonsin mouse systems; Outchkourov (2002) Protein Expr. Purif. 24:18-24,describing optimizing codons in yeast; Feng (2000) Biochemistry39:15399-15409, describing optimizing codons in E. coli; Humphreys(2000) Protein Expr. Purif. 20:252-264, describing optimizing codonusage that affects secretion in E. coli.

Transgenic Non-Human Animals

The invention provides transgenic non-human animals comprising a nucleicacid, a polypeptide (e.g., an isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase), an expressioncassette or vector or a transfected or transformed cell of theinvention. The invention also provides methods of making and using thesetransgenic non-human animals.

The transgenic non-human animals can be, e.g., goats, rabbits, sheep,pigs, cows, rats, horses, dogs, fish and mice, comprising the nucleicacids of the invention. These animals can be used, e.g., as in vivomodels to study isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase activity, or, as models to screen foragents that change the isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase activity in vivo. Thecoding sequences for the polypeptides to be expressed in the transgenicnon-human animals can be designed to be constitutive, or, under thecontrol of tissue-specific, developmental-specific or inducibletranscriptional regulatory factors. Transgenic non-human animals can bedesigned and generated using any method known in the art; see, e.g.,U.S. Pat. Nos. 6,211,428; 6,187,992; 6,156,952; 6,118,044; 6,111,166;6,107,541; 5,959,171; 5,922,854; 5,892,070; 5,880,327; 5,891,698;5,639,940; 5,573,933; 5,387,742; 5,087,571, describing making and usingtransformed cells and eggs and transgenic mice, rats, rabbits, sheep,pigs, chickens, goats, fish and cows. See also, e.g., Pollock (1999) J.Immunol. Methods 231:147-157, describing the production of recombinantproteins in the milk of transgenic dairy animals; Baguisi (1999) Nat.Biotechnol. 17:456-461, demonstrating the production of transgenicgoats. U.S. Pat. No. 6,211,428, describes making and using transgenicnon-human mammals which express in their brains a nucleic acid constructcomprising a DNA sequence. U.S. Pat. No. 5,387,742, describes injectingcloned recombinant or synthetic DNA sequences into fertilized mouseeggs, implanting the injected eggs in pseudo-pregnant females, andgrowing to term transgenic mice whose cells express proteins related tothe pathology of Alzheimer's disease. U.S. Pat. No. 6,187,992, describesmaking and using a transgenic mouse whose genome comprises a disruptionof the gene encoding amyloid precursor protein (APP).

“Knockout animals” can also be used to practice the methods of theinvention. For example, in one aspect, the transgenic or modifiedanimals of the invention comprise a “knockout animal,” e.g., a “knockoutmouse,” engineered not to express an endogenous gene, which is replacedwith a gene expressing an isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase of the invention, or, afusion protein comprising an isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase of the invention.

Transgenic Plants and Seeds

The invention provides transgenic plants and seeds comprising a nucleicacid, a polypeptide (e.g., an isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase), an expressioncassette or vector or a transfected or transformed cell of theinvention. The invention also provides plant products or byproducts,e.g., fruits, oils, seeds, leaves, extracts and the like, including anyplant part, comprising a nucleic acid and/or a polypeptide (e.g., anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase) of the invention, e.g., wherein the nucleic acid orpolypeptide of the invention is heterologous to the plant, plant part,seed etc. The transgenic plant (which includes plant parts, fruits,seeds etc.) can be dicotyledonous (a dicot) or monocotyledonous (amonocot). The invention also provides methods of making and using thesetransgenic plants and seeds. The transgenic plant or plant cellexpressing a polypeptide of the present invention may be constructed inaccordance with any method known in the art. See, for example, U.S. Pat.No. 6,309,872.

Nucleic acids and expression constructs of the invention can beintroduced into a plant cell by any means. For example, nucleic acids orexpression constructs can be introduced into the genome of a desiredplant host, or, the nucleic acids or expression constructs can beepisomes. Introduction into the genome of a desired plant can be suchthat the host's isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase production is regulated by endogenoustranscriptional or translational control elements. The invention alsoprovides “knockout plants” where insertion of gene sequence by, e.g.,homologous recombination, has disrupted the expression of the endogenousgene. Means to generate “knockout” plants are well-known in the art,see, e.g., Strepp (1998) Proc Natl. Acad. Sci. USA 95:4368-4373; Miao(1995) Plant J 7:359-365. See discussion on transgenic plants, below.

The nucleic acids of the invention can be used to confer desired traitson essentially any plant, e.g., on starch-producing plants, such aspotato, wheat, rice, barley, and the like. Nucleic acids of theinvention can be used to manipulate metabolic pathways of a plant inorder to optimize or alter host's expression of an isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerase.The can change isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase activity in a plant. Alternatively,an isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase of the invention can be used in production ofa transgenic plant to produce a compound not naturally produced by thatplant. This can lower production costs or create a novel product.

In one aspect, the first step in production of a transgenic plantinvolves making an expression construct for expression in a plant cell.These techniques are well known in the art. They can include selectingand cloning a promoter, a coding sequence for facilitating efficientbinding of ribosomes to mRNA and selecting the appropriate geneterminator sequences. One exemplary constitutive promoter is CaMV35S,from the cauliflower mosaic virus, which generally results in a highdegree of expression in plants. Other promoters are more specific andrespond to cues in the plant's internal or external environment. Anexemplary light-inducible promoter is the promoter from the cab gene,encoding the major chlorophyll a/b binding protein.

In one aspect, the nucleic acid is modified to achieve greaterexpression in a plant cell. For example, a sequence of the invention islikely to have a higher percentage of A-T nucleotide pairs compared tothat seen in a plant, some of which prefer G-C nucleotide pairs.Therefore, A-T nucleotides in the coding sequence can be substitutedwith G-C nucleotides without significantly changing the amino acidsequence to enhance production of the gene product in plant cells.

Selectable marker gene can be added to the gene construct in order toidentify plant cells or tissues that have successfully integrated thetransgene. This may be necessary because achieving incorporation andexpression of genes in plant cells is a rare event, occurring in just afew percent of the targeted tissues or cells. Selectable marker genesencode proteins that provide resistance to agents that are normallytoxic to plants, such as antibiotics or herbicides. Only plant cellsthat have integrated the selectable marker gene will survive when grownon a medium containing the appropriate antibiotic or herbicide. As forother inserted genes, marker genes also require promoter and terminationsequences for proper function.

In one aspect, making transgenic plants or seeds comprises incorporatingsequences of the invention and, in one aspect (optionally), marker genesinto a target expression construct (e.g., a plasmid), along withpositioning of the promoter and the terminator sequences. This caninvolve transferring the modified gene into the plant through a suitablemethod. For example, a construct may be introduced directly into thegenomic DNA of the plant cell using techniques such as electroporationand microinjection of plant cell protoplasts, or the constructs can beintroduced directly to plant tissue using ballistic methods, such as DNAparticle bombardment. For example, see, e.g., Christou (1997) Plant Mol.Biol. 35:197-203; Pawlowski (1996) Mol. Biotechnol. 6:17-30; Klein(1987) Nature 327:70-73; Takumi (1997) Genes Genet. Syst. 72:63-69,discussing use of particle bombardment to introduce transgenes intowheat; and Adam (1997) supra, for use of particle bombardment tointroduce YACs into plant cells. For example, Rinehart (1997) supra,used particle bombardment to generate transgenic cotton plants.Apparatus for accelerating particles is described U.S. Pat. No.5,015,580; and, the commercially available BioRad (Biolistics) PDS-2000particle acceleration instrument; see also, John, U.S. Pat. No.5,608,148; and Ellis, U.S. Pat. No. 5,681,730, describingparticle-mediated transformation of gymnosperms.

In one aspect, protoplasts can be immobilized and injected with anucleic acids, e.g., an expression construct. Although plantregeneration from protoplasts is not easy with cereals, plantregeneration is possible in legumes using somatic embryogenesis fromprotoplast derived callus. Organized tissues can be transformed withnaked DNA using gene gun technique, where DNA is coated on tungstenmicroprojectiles, shot 1/100th the size of cells, which carry the DNAdeep into cells and organelles. Transformed tissue is then induced toregenerate, usually by somatic embryogenesis. This technique has beensuccessful in several cereal species including maize and rice.

Nucleic acids, e.g., expression constructs, can also be introduced in toplant cells using recombinant viruses. Plant cells can be transformedusing viral vectors, such as, e.g., tobacco mosaic virus derived vectors(Rouwendal (1997) Plant Mol. Biol. 33:989-999), see Porta (1996) “Use ofviral replicons for the expression of genes in plants,” Mol. Biotechnol.5:209-221.

Alternatively, nucleic acids, e.g., an expression construct, can becombined with suitable T-DNA flanking regions and introduced into aconventional Agrobacterium tumefaciens host vector. The virulencefunctions of the Agrobacterium tumefaciens host will direct theinsertion of the construct and adjacent marker into the plant cell DNAwhen the cell is infected by the bacteria. Agrobacteriumtumefaciens-mediated transformation techniques, including disarming anduse of binary vectors, are well described in the scientific literature.See, e.g., Horsch (1984) Science 233:496-498; Fraley (1983) Proc. Natl.Acad. Sci. USA 80:4803 (1983); Gene Transfer to Plants, Potrykus, ed.(Springer-Verlag, Berlin 1995). The DNA in an A. tumefaciens cell iscontained in the bacterial chromosome as well as in another structureknown as a Ti (tumor-inducing) plasmid. The Ti plasmid contains astretch of DNA termed T-DNA (˜20 kb long) that is transferred to theplant cell in the infection process and a series of vir (virulence)genes that direct the infection process. A. tumefaciens can only infecta plant through wounds: when a plant root or stem is wounded it givesoff certain chemical signals, in response to which, the vir genes of A.tumefaciens become activated and direct a series of events necessary forthe transfer of the T-DNA from the Ti plasmid to the plant's chromosome.The T-DNA then enters the plant cell through the wound. One speculationis that the T-DNA waits until the plant DNA is being replicated ortranscribed, then inserts itself into the exposed plant DNA. In order touse A. tumefaciens as a transgene vector, the tumor-inducing section ofT-DNA have to be removed, while retaining the T-DNA border regions andthe vir genes. The transgene is then inserted between the T-DNA borderregions, where it is transferred to the plant cell and becomesintegrated into the plant's chromosomes.

The invention provides for the transformation of monocotyledonous plantsusing the nucleic acids of the invention, including important cereals,see Hiei (1997) Plant Mol. Biol. 35:205-218. See also, e.g., Horsch,Science (1984) 233:496; Fraley (1983) Proc. Natl. Acad. Sci USA 80:4803;Thykjaer (1997) supra; Park (1996) Plant Mol. Biol. 32:1135-1148,discussing T-DNA integration into genomic DNA. See also D'Halluin, U.S.Pat. No. 5,712,135, describing a process for the stable integration of aDNA comprising a gene that is functional in a cell of a cereal, or othermonocotyledonous plant.

In one aspect, the third step can involve selection and regeneration ofwhole plants capable of transmitting the incorporated target gene to thenext generation. Such regeneration techniques rely on manipulation ofcertain phytohormones in a tissue culture growth medium, typicallyrelying on a biocide and/or herbicide marker that has been introducedtogether with the desired nucleotide sequences. Plant regeneration fromcultured protoplasts is described in Evans et al., Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp. 124-176, MacMillilanPublishing Company, New York, 1983; and Binding, Regeneration of Plants,Plant Protoplasts, pp. 21-73, CRC Press, Boca Raton, 1985. Regenerationcan also be obtained from plant callus, explants, organs, or partsthereof. Such regeneration techniques are described generally in Klee(1987) Ann. Rev. of Plant Phys. 38:467-486. To obtain whole plants fromtransgenic tissues such as immature embryos, they can be grown undercontrolled environmental conditions in a series of media containingnutrients and hormones, a process known as tissue culture. Once wholeplants are generated and produce seed, evaluation of the progeny begins.

After the expression cassette is stably incorporated in transgenicplants, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed. Since transgenic expression of the nucleicacids of the invention leads to phenotypic changes, plants comprisingthe recombinant nucleic acids of the invention can be sexually crossedwith a second plant to obtain a final product. Thus, the seed of theinvention can be derived from a cross between two transgenic plants ofthe invention, or a cross between a plant of the invention and anotherplant. The desired effects (e.g., expression of the polypeptides of theinvention to produce a plant in which flowering behavior is altered) canbe enhanced when both parental plants express the polypeptides (e.g., anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase) of the invention. The desired effects can be passed tofuture plant generations by standard propagation means.

The nucleic acids and polypeptides of the invention are expressed in orinserted in any plant or seed. Transgenic plants of the invention can bedicotyledonous or monocotyledonous. Examples of monocot transgenicplants of the invention are grasses, such as meadow grass (blue grass,Poa), forage grass such as festuca, lolium, temperate grass, such asAgrostis, and cereals, e.g., wheat, oats, rye, barley, rice, sorghum,and maize (corn). Examples of dicot transgenic plants of the inventionare tobacco, legumes, such as lupins, potato, sugar beet, pea, bean andsoybean, and cruciferous plants (family Brassicaceae), such ascauliflower, rape seed, and the closely related model organismArabidopsis thaliana. Thus, the transgenic plants and seeds of theinvention include a broad range of plants, including, but not limitedto, species from the genera Anacardium, Arachis, Asparagus, Atropa,Avena, Brassica, Citrus, Citrullus, Capsicum, Carthamus, Cocos, Coffea,Cucumis, Cucurbita, Daucus, Elaeis, Fragaria, Glycine, Gossypium,Helianthus, Heterocallis, Hordeum, Hyoscyamus, Lactuca, Linum, Lolium,Lupinus, Lycopersicon, Malus, Manihot, Majorana, Medicago, Nicotiana,Olea, Oryza, Panieum, Pannisetum, Persea, Phaseolus, Pistachia, Pisum,Pyrus, Prunus, Raphanus, Ricinus, Secale, Senecio, Sinapis, Solanum,Sorghum, Theobromus, Trigonella, Triticum, Vicia, Vitis, Vigna, and Zea.Transgenic plants and seeds of the invention can be any monocot ordicot, e.g., a monocot corn, sugarcane, rice, wheat, barley, switchgrassor Miscanthus; or a dicot oilseed crop, soy, canola, rapeseed, flax,cotton, palm oil, sugar beet, peanut, tree, poplar or lupine.

In alternative embodiments, the nucleic acids of the invention areexpressed in plants (and/or their seeds) which contain fiber cells,including, e.g., cotton, silk cotton tree (Kapok, Ceiba pentandra),desert willow, creosote bush, winterfat, balsa, ramie, kenaf, hemp,roselle, jute, sisal abaca and flax. In alternative embodiments, thetransgenic plants of the invention can be members of the genusGossypium, including members of any Gossypium species, such as G.arboreum; G. herbaceum, G. barbadense, and G. hirsutum.

The invention also provides for transgenic plants (and/or their seeds)to be used for producing large amounts of the polypeptides (e.g., anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase or antibody) of the invention. For example, seePalmgren (1997) Trends Genet. 13:348; Chong (1997) Transgenic Res.6:289-296 (producing human milk protein beta-casein in transgenic potatoplants using an auxin-inducible, bidirectional mannopine synthase(mas1′,2′) promoter with Agrobacterium tumefaciens-mediated leaf disctransformation methods).

Using known procedures, one of skill can screen for plants (and/or theirseeds) of the invention by detecting the increase or decrease oftransgene mRNA or protein in transgenic plants. Means for detecting andquantitation of mRNAs or proteins are well known in the art.

Polypeptides and Peptides

In one aspect, the invention provides isolated, synthetic or recombinantpolypeptides and peptides having isomerase activity, e.g., racemaseactivity, e.g., amino acid racemase activity (e.g., resolving a D-and/or an L-amino acid from a racemic mixture), alanine racemaseactivity, and/or epimerase activity, or polypeptides and peptidescapable of generating an antibody that specifically binds to anisomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase, including an enzyme of this invention,including the amino acid sequences of the invention, which include thosehaving at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%,61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more, or 100%(complete) sequence identity to an exemplary polypeptide of theinvention (as defined above, including SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ IDNO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ IDNO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:44, SEQ IDNO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ ID NO:54, SEQ IDNO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, SEQ IDNO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ ID NO:74, SEQ IDNO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ ID NO:84, SEQ IDNO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ ID NO:94, SEQ IDNO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:104, SEQ IDNO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQ ID NO:114, SEQID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124,SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ IDNO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150, SEQ ID NO:152,SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ ID NO:160, SEQ IDNO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQ ID NO:170, SEQID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178, SEQ ID NO:180,SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ ID NO:188, SEQ IDNO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQ ID NO:198, SEQID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206, SEQ ID NO:208,SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ ID NO:216, SEQ IDNO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQ ID NO:226, SEQID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234, SEQ ID NO:236,SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ ID NO:244, SEQ IDNO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQ ID NO:254, SEQID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264,SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ ID NO:272, SEQ IDNO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQ ID NO:282, SEQID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290, SEQ ID NO:292,SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ ID NO:300, SEQ IDNO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQ ID NO:310, SEQID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318, SEQ ID NO:320,SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ ID NO:328, SEQ IDNO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQ ID NO:338, SEQID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346, SEQ ID NO:348,SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ ID NO:356, SEQ IDNO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQ ID NO:366, SEQID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374, SEQ ID NO:376,SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ ID NO:384, SEQ IDNO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ ID NO:394, SEQID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQ ID NO:404,SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ ID NO:412, SEQ IDNO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ ID NO:422, SEQID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430, SEQ ID NO:432,SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ ID NO:440, SEQ IDNO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460,SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ IDNO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQ ID NO:478, SEQID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486, SEQ ID NO:488,SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ ID NO:496 or SEQ IDNO:498), including the sequences described herein and in Tables 1, 2 and3, and the Sequence Listing (all of these sequences are “exemplaryenzymes/polypeptides of the invention”), and enzymatically activesubsequences (fragments) thereof.

In one aspect, the invention provides chimeric enzymes, including anisomerase, e.g., a racemase, e.g., an amino acid racemase, an alanineracemase, and/or an epimerase, having heterologous domains, e.g., abinding domain or a dockerin domain, e.g., for use in the processes ofthe invention and in various industrial, medical, pharmaceutical,research, food and feed and food and feed supplement processing andother applications. For example, in one aspect the invention providesenzymes, e.g., isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases comprising one or more domain of anenzyme of the invention. In another aspect, domains between differentenzymes of the invention can be swapped; or, alternatively, one or moredomains of one or more enzymes of the invention can be spliced into anenzyme, e.g., an isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase. In one aspect of the invention, thedomains are selected from a binding domain or a dockerin domain.

The invention further provides chimeric enzymes having heterologous,non-natural substrates; including chimeric enzymes having multiplesubstrates by nature of their “spliced-in” heterologous domains—thusgiving the chimeric enzyme new specificity or enhanced binding. Theheterologous domains of the chimeric enzymes of the invention can bedesigned to be modular, i.e., to be appended to a catalytic module orcatalytic domain (e.g., an active site), which also can be heterologousor can be homologous to the enzyme.

Utilization of just the catalytic module of an isomerase, e.g., aracemase, e.g., an amino acid racemase, an alanine racemase, and/or anepimerase (e.g., an enzyme of the invention) has been shown to beeffective. Thus, the invention provides peptides and polypeptidesconsisting of, or comprising, modular domains/active site modules, whichcan be homologously paired or joined as chimeric (heterologous) activesite module pairs. Thus, these chimeric polypeptides/peptides of theinvention can be used to improve or alter the performance of anindividual enzyme, e.g., an isomerase, e.g., a racemase, e.g., an aminoacid racemase, an alanine racemase, and/or an epimerase enzyme. Achimeric catalytic module of the invention (comprising, e.g., at leastone domain of the invention) can be designed to target the enzyme toparticular regions of a substrate. For example, in one aspect, this isachieved by making fusions of the isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase and various domains(either an isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase of the invention with a heterologous domain,or, a domain of the invention with another enzyme, e.g., an isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase.

Thus, the invention provides chimeric isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases, e.g., afusion of an isomerase, e.g., a racemase, e.g., an amino acid racemase,an alanine racemase, and/or an epimerase with different (e.g.,heterologous) domains. In one aspect, the chimeric isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases comprise an enzyme of the invention. In one aspect, thechimeric enzyme comprises fusions of different domains. The inventionalso provides methods comprising recombining different domains withdifferent isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases (e.g., domains of the inventionand/or isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention) and screening theresultant chimerics to find the best combination for a particularapplication or substrate.

Other variations also are within the scope of this invention, e.g.,where one, two, three, four or five or more residues are removed fromthe carboxy- or amino-terminal ends of any polypeptide of the invention.Another variation includes modifying any residue to increase or decreasepI of a polypeptide, e.g., removing or modifying (e.g., to another aminoacid) a glutamate. This method was used as a general scheme forimproving the enzyme's properties without creating regulatory issuessince no amino acids are mutated; and this general scheme can be usedwith any polypeptide of the invention.

The invention provides isolated, synthetic or recombinant polypeptideshaving isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase activity, wherein the polypeptide has asequence modification of any polypeptide of the invention, including anyexemplary amino acid sequence of the invention, including SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ IDNO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:42, SEQ IDNO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NO:50, SEQ ID NO:52, SEQ IDNO:54, SEQ ID NO:56, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ IDNO:64, SEQ ID NO:66, SEQ ID NO:68, SEQ ID NO:70, SEQ ID NO:72, SEQ IDNO:74, SEQ ID NO:76, SEQ ID NO:78, SEQ ID NO:80, SEQ ID NO:82, SEQ IDNO:84, SEQ ID NO:86, SEQ ID NO:88, SEQ ID NO:90, SEQ ID NO:92, SEQ IDNO:94, SEQ ID NO:96, SEQ ID NO:98, SEQ ID NO:100, SEQ ID NO:102, SEQ IDNO:104, SEQ ID NO:106, SEQ ID NO:108, SEQ ID NO:110, SEQ ID NO:112, SEQID NO:114, SEQ ID NO:116, SEQ ID NO:118, SEQ ID NO:120, SEQ ID NO:122,SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ IDNO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQID NO:142, SEQ ID NO:143, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:150,SEQ ID NO:152, SEQ ID NO:154, SEQ ID NO:156, SEQ ID NO:158, SEQ IDNO:160, SEQ ID NO:162, SEQ ID NO:164, SEQ ID NO:166, SEQ ID NO:168, SEQID NO:170, SEQ ID NO:172, SEQ ID NO:174, SEQ ID NO:176, SEQ ID NO:178,SEQ ID NO:180, SEQ ID NO:182, SEQ ID NO:184, SEQ ID NO:186, SEQ IDNO:188, SEQ ID NO:190, SEQ ID NO:192, SEQ ID NO:194, SEQ ID NO:196, SEQID NO:198, SEQ ID NO:200, SEQ ID NO:202, SEQ ID NO:204, SEQ ID NO:206,SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:212, SEQ ID NO:214, SEQ IDNO:216, SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, SEQ ID NO:224, SEQID NO:226, SEQ ID NO:228, SEQ ID NO:230, SEQ ID NO:232, SEQ ID NO:234,SEQ ID NO:236, SEQ ID NO:238, SEQ ID NO:240, SEQ ID NO:242, SEQ IDNO:244, SEQ ID NO:246, SEQ ID NO:248, SEQ ID NO:250, SEQ ID NO:252, SEQID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262,SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, SEQ ID NO:270, SEQ IDNO:272, SEQ ID NO:274, SEQ ID NO:276, SEQ ID NO:278, SEQ ID NO:280, SEQID NO:282, SEQ ID NO:284, SEQ ID NO:286, SEQ ID NO:288, SEQ ID NO:290,SEQ ID NO:292, SEQ ID NO:294, SEQ ID NO:296, SEQ ID NO:298, SEQ IDNO:300, SEQ ID NO:302, SEQ ID NO:304, SEQ ID NO:306, SEQ ID NO:308, SEQID NO:310, SEQ ID NO:312, SEQ ID NO:314, SEQ ID NO:316, SEQ ID NO:318,SEQ ID NO:320, SEQ ID NO:322, SEQ ID NO:324, SEQ ID NO:326, SEQ IDNO:328, SEQ ID NO:330, SEQ ID NO:332, SEQ ID NO:334, SEQ ID NO:336, SEQID NO:338, SEQ ID NO:340, SEQ ID NO:342, SEQ ID NO:344, SEQ ID NO:346,SEQ ID NO:348, SEQ ID NO:350, SEQ ID NO:352, SEQ ID NO:354, SEQ IDNO:356, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, SEQ ID NO:364, SEQID NO:366, SEQ ID NO:368, SEQ ID NO:370, SEQ ID NO:372, SEQ ID NO:374,SEQ ID NO:376, SEQ ID NO:378, SEQ ID NO:380, SEQ ID NO:382, SEQ IDNO:384, SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQID NO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402,SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408, SEQ ID NO:410, SEQ IDNO:412, SEQ ID NO:414, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQID NO:422, SEQ ID NO:424, SEQ ID NO:426, SEQ ID NO:428, SEQ ID NO:430,SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438, SEQ IDNO:440, SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQID NO:450, SEQ ID NO:452, SEQ ID NO:454, SEQ ID NO:456, SEQ ID NO:458,SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ IDNO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476, SEQID NO:478, SEQ ID NO:480, SEQ ID NO:482, SEQ ID NO:484, SEQ ID NO:486,SEQ ID NO:488, SEQ ID NO:490, SEQ ID NO:492, SEQ ID NO:494, SEQ IDNO:496 or SEQ ID NO:498, including the sequences described herein and inTables 1, 2 and 3, and the Sequence Listing (all of these sequences are“exemplary enzymes/polypeptides of the invention”), and enzymaticallyactive subsequences (fragments) thereof. The sequence change(s) can alsocomprise any amino acid modification to change the pI of a polypeptide,e.g., deletion or modification of a glutamate, or changing from aglutamate to another residue.

The invention further provides isolated, synthetic or recombinantpolypeptides having a sequence identity (e.g., at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99%, or more, or complete (100%) sequenceidentity) to an exemplary sequence of the invention.

In one aspect, the polypeptide of the invention has an isomeraseactivity, e.g., racemase activity, e.g., amino acid racemase activity,alanine racemase activity, and/or epimerase activity, and/or catalyzethe re-arrangement of atoms within a molecule, catalyze the conversionof one isomer into another, catalyze the conversion of an opticallyactive substrate into a raceme, which is optically inactive, catalyzethe interconversion of substrate enantiomers, catalyze thestereochemical inversion around the asymmetric carbon atom in asubstrate having only one center of asymmetry, catalyze thestereochemical inversion of the configuration around an asymmetriccarbon atom in a substrate having more than one asymmetric center,and/or catalyze the racemization of amino acids.

In one aspect, a polypeptide of the invention has a 5-substitutedhydantoin racemase activity, e.g., it can catalyze racemization reactionof an optically active 5-substituted hydantoin compound, such as a D- orL-5-substituted hydantoin compound.

In one aspect, a polypeptide of the invention has peptidyl-prolylisomerase activity, and can be part of a signaling pathway that leads toT-cell activation, and/or correct protein folding and/or proteintrafficking, and also can be involved in assembly/disassembly of proteincomplexes and regulation of protein activity.

In one aspect, a polypeptide of the invention has a racemase, orisomerase, activity that catalyzes inversion of a molecule'sconfiguration around an asymmetric carbon atom in a substrate having asingle center of asymmetry, thereby interconverting two racemers.

In one aspect, a polypeptide of the invention has a racemase, or anepimerase activity, that catalyzes inversion of configuration around anasymmetric carbon atom in a substrate with more than one center ofsymmetry, thereby interconverting two epimers. Racemases and epimerasesof this invention can act on amino acids and their derivatives, hydroxyacids and their derivatives, and carbohydrates and their derivatives.For example, the interconversion of UDP-galactose and UDP-glucose can becatalyzed by an enzyme of this invention having aUDP-galactose-4′-epimerase activity; proper regulation and function ofthis epimerase is essential to the synthesis of glycoproteins andglycolipids. Elevated blood galactose levels have been correlated withUDP-galactose-4′-epimerase deficiency in screening programs of infants.

In one aspect, a polypeptide of the invention has a serine racemaseenzyme activity, and this enzyme can be used to increase or decreaseD-serine formation, which can be used as a pharmaceutical (drug) in anindividual, e.g., to increase or decrease NMDA receptor activation; adecrease in D-serine formation (by using a serine racemase enzyme ofthis invention) can aid in the prevention of neuron damage following anischemic event, such as stroke; and regulation of D-serine formation bya serine racemase enzyme of this invention also can be effective fortreating a neurodegenerative condition caused by the over- orunder-activation of the glutamate NMDA receptor.

Polypeptides of the invention having a serine racemase enzyme activitycan be used to regulate D-serine levels, e.g., to prevent or minimizeneuron damage caused, for example, by primary and/or secondary disordersafter brain injury, motor unit-like neurogenic and myopathic disorders,neurodegenerative disorders like Alzheimer's and Parkinson's disease,disorders leading to peripheral and chronic pain. See, e.g., U.S. Pat.App. Pub. No. 20030175941.

Any isomerase activity, e.g., racemase activity, e.g., amino acidracemase activity, alanine racemase activity, and/or epimerase activityassay known in the art can be used to determine if a polypeptide hasisomerase activity, e.g., racemase activity, e.g., amino acid racemaseactivity, alanine racemase activity, and/or epimerase activity and iswithin scope of the invention. For example, Schonfeld and Bomscheuer(Anal Chem. 2004 Feb. 15; 76(4):1184-8) describe a polarimetric assaywhich identifies alpha-amino acid racemase activity using a glutamateracemase from Lactobacillus fermentii, expressed in E. coli, andmeasuring the time-dependent change of the optical rotation using the1-glutamate as the substrate.

Another exemplary method for detecting racemase activity to determine ifa polypeptide is within the scope of this invention is described in U.S.Pat. App. Pub. No. 20070128601, and comprises providing a reactionmedium containing a D-amino acid specific to the racemase to bedetected; reacting the D-amino acid with a D-amino oxidase with aprosthetic group to form a reduced prosthetic group by oxidation of theD-amino acid; reacting the reduced prosthetic group with oxygen to formhydrogen peroxide; and detecting the hydrogen peroxide thus formed;wherein the detection of hydrogen peroxide indicates racemase activityin the reaction medium. An exemplary method for detecting a D-amino acidin a sample comprises oxidatively deaminating a D-amino acid by reactionwith a D-amino acid oxidase in a prosthetic group; and, detecting thehydrogen peroxide generated by the oxidative deamination; wherein thepresence of hydrogen peroxide is indicative of the presence of a D-aminoacid in the sample.

Another exemplary method for detecting racemase activity to determine ifa polypeptide is within the scope of this invention is described in U.S.Pat. App. Pub. No. 20060014162, and comprises detecting a D-amino acidby providing a reaction medium containing a D-amino acid; reacting theD-amino acid with a D-amino oxidase with a prosthetic group to form areduced prosthetic group by oxidative deamination of the D-amino acidwith a primary amine or oxidation of the D-amino acid with a secondaryamine; reacting the reduced prosthetic group with oxygen to formhydrogen peroxide; and detecting the hydrogen peroxide thus formed.

The polypeptides of the invention include isomerases, e.g., racemases,e.g., amino acid racemases, alanine racemases, and/or epimerases in anactive or inactive form. For example, the polypeptides of the inventioninclude proproteins before “maturation” or processing of preprosequences, e.g., by a proprotein-processing enzyme, such as a proproteinconvertase to generate an “active” mature protein. The polypeptides ofthe invention include isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases inactive for otherreasons, e.g., before “activation” by a post-translational processingevent, e.g., an endo- or exo-peptidase or proteinase action, aphosphorylation event, an amidation, a glycosylation or a sulfation, adimerization event, and the like. The polypeptides of the inventioninclude all active forms, including active subsequences, e.g., catalyticdomains or active sites, of the isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase.

Methods for identifying “prepro” domain sequences and signal sequencesare well known in the art, see, e.g., Van de Ven (1993) Crit. Rev.Oncog. 4(2):115-136. For example, to identify a prepro sequence, theprotein is purified from the extracellular space and the N-terminalprotein sequence is determined and compared to the unprocessed form.

The invention includes polypeptides with or without a signal sequenceand/or a prepro sequence. The invention includes polypeptides withheterologous signal sequences and/or prepro sequences. The preprosequence (including a sequence of the invention used as a heterologousprepro domain) can be located on the amino terminal or the carboxyterminal end of the protein. The invention also includes isolated,synthetic or recombinant signal sequences, prepro sequences andcatalytic domains (e.g., “active sites”) comprising sequences of theinvention.

The percent sequence identity can be over the full length of thepolypeptide, or, the identity can be over a region of at least about 50,60, 70, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700 or more residues. Polypeptides of the invention can also beshorter than the full length of exemplary polypeptides. In alternativeaspects, the invention provides polypeptides (peptides, fragments)ranging in size between about 5 and the full length of a polypeptide,e.g., an enzyme, such as an isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase; exemplary sizes being ofabout 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, 500, 550, 600,650, 700, or more residues, e.g., contiguous residues of an exemplaryisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase of the invention.

Peptides of the invention (e.g., a subsequence of an exemplarypolypeptide of the invention) can be useful as, e.g., labeling probes,antigens, toleragens, motifs, active sites (e.g., “catalytic domains”),signal sequences and/or prepro domains.

Polypeptides and peptides of the invention can be isolated from naturalsources, be synthetic, or be recombinantly generated polypeptides.Peptides and proteins can be recombinantly expressed in vitro or invivo. The peptides and polypeptides of the invention can be made andisolated using any method known in the art. Polypeptides and peptides ofthe invention can also be synthesized, whole or in part, using chemicalmethods well known in the art. See e.g., Caruthers (1980) Nucleic AcidsRes. Symp. Ser. 215-223; Horn (1980) Nucleic Acids Res. Symp. Ser.225-232; Banga, A. K., Therapeutic Peptides and Proteins, Formulation,Processing and Delivery Systems (1995) Technomic Publishing Co.,Lancaster, Pa. For example, peptide synthesis can be performed usingvarious solid-phase techniques (see e.g., Roberge (1995) Science269:202; Merrifield (1997) Methods Enzymol. 289:3-13) and automatedsynthesis may be achieved, e.g., using the ABI 431A Peptide Synthesizer(Perkin Elmer) in accordance with the instructions provided by themanufacturer.

The peptides and polypeptides of the invention can also be glycosylated.The glycosylation can be added post-translationally either chemically orby cellular biosynthetic mechanisms, wherein the later incorporates theuse of known glycosylation motifs, which can be native to the sequenceor can be added as a peptide or added in the nucleic acid codingsequence. The glycosylation can be O-linked or N-linked.

“Amino acid” or “amino acid sequence” as used herein refer to anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these and to naturally occurringor synthetic molecules. “Amino acid” or “amino acid sequence” include anoligopeptide, peptide, polypeptide, or protein sequence, or to afragment, portion, or subunit of any of these, and to naturallyoccurring or synthetic molecules. The term “polypeptide” as used herein,refers to amino acids joined to each other by peptide bonds or modifiedpeptide bonds, i.e., peptide isosteres and may contain modified aminoacids other than the 20 gene-encoded amino acids. The polypeptides maybe modified by either natural processes, such as post-translationalprocessing, or by chemical modification techniques that are well knownin the art. Modifications can occur anywhere in the polypeptide,including the peptide backbone, the amino acid side-chains and the aminoor carboxyl termini. It will be appreciated that the same type ofmodification may be present in the same or varying degrees at severalsites in a given polypeptide. Also a given polypeptide may have manytypes of modifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, phosphorylation, prenylation, racemization, selenoylation,sulfation and transfer-RNA mediated addition of amino acids to proteinsuch as arginylation. (See Creighton, T. E., Proteins—Structure andMolecular Properties 2nd Ed., W.H. Freeman and Company, New York (1993);Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York, pp. 1-12 (1983)). The peptides andpolypeptides of the invention also include all “mimetic” and“peptidomimetic” forms, as described in further detail, below.

“Recombinant” polypeptides or proteins refer to polypeptides or proteinsproduced by recombinant DNA techniques; i.e., produced from cellstransformed by an exogenous DNA construct encoding the desiredpolypeptide or protein. “Synthetic” nucleic acids (includingoligonucleotides), polypeptides or proteins of the invention includethose prepared by any chemical synthesis, e.g., as described, below.Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

“Fragments” as used herein are a portion of a naturally occurringprotein which can exist in at least two different conformations.Fragments can have the same or substantially the same amino acidsequence as the naturally occurring protein. “Substantially the same”means that an amino acid sequence is largely, but not entirely, thesame, but retains at least one functional activity of the sequence towhich it is related. In general two amino acid sequences are“substantially the same” or “substantially homologous” if they are atleast about 85% identical. Fragments which have different threedimensional structures as the naturally occurring protein are alsoincluded. An example of this, is a “pro-form” molecule, such as a lowactivity proprotein that can be modified by cleavage to produce a matureenzyme with significantly higher activity.

The peptides and polypeptides of the invention, as defined above,include all “mimetic” and “peptidomimetic” forms. The terms “mimetic”and “peptidomimetic” refer to a synthetic chemical compound which hassubstantially the same structural and/or functional characteristics ofthe polypeptides of the invention. The mimetic can be either entirelycomposed of synthetic, non-natural analogues of amino acids, or, is achimeric molecule of partly natural peptide amino acids and partlynon-natural analogs of amino acids. The mimetic can also incorporate anyamount of natural amino acid conservative substitutions as long as suchsubstitutions also do not substantially alter the mimetic's structureand/or activity. As with polypeptides of the invention which areconservative variants, routine experimentation will determine whether amimetic is within the scope of the invention, i.e., that its structureand/or function is not substantially altered. Thus, in one aspect, amimetic composition is within the scope of the invention if it has anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase activity.

Polypeptide mimetic compositions of the invention can contain anycombination of non-natural structural components. In alternative aspect,mimetic compositions of the invention include one or all of thefollowing three structural groups: a) residue linkage groups other thanthe natural amide bond (“peptide bond”) linkages; b) non-naturalresidues in place of naturally occurring amino acid residues; or c)residues which induce secondary structural mimicry, i.e., to induce orstabilize a secondary structure, e.g., a beta turn, gamma turn, betasheet, alpha helix conformation, and the like. For example, apolypeptide of the invention can be characterized as a mimetic when allor some of its residues are joined by chemical means other than naturalpeptide bonds. Individual peptidomimetic residues can be joined bypeptide bonds, other chemical bonds or coupling means, such as, e.g.,glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides,N,N′-dicyclohexylcarbodiimide (DCC) or N,N′-diisopropylcarbodiimide(DIC). Linking groups that can be an alternative to the traditionalamide bond (“peptide bond”) linkages include, e.g., ketomethylene (e.g.,—C(═O)—CH₂— for —C(═O)—NH—), aminomethylene (CH₂—NH), ethylene, olefin(CH═CH), ether (CH₂—O), thioether (CH₂—S), tetrazole (CN₄—), thiazole,retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistryand Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp267-357, “Peptide Backbone Modifications,” Marcell Dekker, NY).

A polypeptide of the invention can also be characterized as a mimetic bycontaining all or some non-natural residues in place of naturallyoccurring amino acid residues. Non-natural residues are well describedin the scientific and patent literature; a few exemplary non-naturalcompositions useful as mimetics of natural amino acid residues andguidelines are described below. Mimetics of aromatic amino acids can begenerated by replacing by, e.g., D- or L-naphylalanine; D- orL-phenylglycine; D- or L-2 thieneylalanine; D- or L-1, -2, 3-, or4-pyreneylalanine; D- or L-3 thieneylalanine; D- orL-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)-alanine; D- orL-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine;D-(trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine;D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; D- orL-p-methoxy-biphenylphenylalanine; D- or L-2-indole(alkyl)alanines; and,D- or L-alkylainines, where alkyl can be substituted or unsubstitutedmethyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl,sec-isotyl, iso-pentyl, or a non-acidic amino acids. Aromatic rings of anon-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl,benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

Mimetics of acidic amino acids can be generated by substitution by,e.g., non-carboxylate amino acids while maintaining a negative charge;(phosphono)alanine; sulfated threonine. Carboxyl side groups (e.g.,aspartyl or glutamyl) can also be selectively modified by reaction withcarbodiimides (R′—N—C—N—R′) such as, e.g.,1-cyclohexyl-3(2-morpholinyl-(4-ethyl)carbodiimide or1-ethyl-3(4-azonia-4,4-dimetholpentyl)carbodiimide. Aspartyl or glutamylcan also be converted to asparaginyl and glutaminyl residues by reactionwith ammonium ions. Mimetics of basic amino acids can be generated bysubstitution with, e.g., (in addition to lysine and arginine) the aminoacids ornithine, citrulline, or (guanidino)-acetic acid, or(guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrilederivative (e.g., containing the CN-moiety in place of COOH) can besubstituted for asparagine or glutamine. Asparaginyl and glutaminylresidues can be deaminated to the corresponding aspartyl or glutamylresidues. Arginine residue mimetics can be generated by reacting arginylwith, e.g., one or more conventional reagents, including, e.g.,phenylglyoxal, 2,3-butanedione, 1,2-cyclo-hexanedione, or ninhydrin,preferably under alkaline conditions. Tyrosine residue mimetics can begenerated by reacting tyrosyl with, e.g., aromatic diazonium compoundsor tetranitromethane. N-acetylimidizol and tetranitromethane can be usedto form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.Cysteine residue mimetics can be generated by reacting cysteinylresidues with, e.g., alpha-haloacetates such as 2-chloroacetic acid orchloroacetamide and corresponding amines; to give carboxymethyl orcarboxyamidomethyl derivatives. Cysteine residue mimetics can also begenerated by reacting cysteinyl residues with, e.g.,bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl)propionic acid;chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide;methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4nitrophenol; or, chloro-7-nitrobenzo-oxa-1,3-diazole. Lysine mimeticscan be generated (and amino terminal residues can be altered) byreacting lysinyl with, e.g., succinic or other carboxylic acidanhydrides. Lysine and other alpha-amino-containing residue mimetics canalso be generated by reaction with imidoesters, such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride,trinitro-benzenesulfonic acid, O-methylisourea, 2,4, pentanedione, andtransamidase-catalyzed reactions with glyoxylate. Mimetics of methioninecan be generated by reaction with, e.g., methionine sulfoxide. Mimeticsof proline include, e.g., pipecolic acid, thiazolidine carboxylic acid,3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or3,3,-dimethylproline. Histidine residue mimetics can be generated byreacting histidyl with, e.g., diethylprocarbonate or para-bromophenacylbromide. Other mimetics include, e.g., those generated by hydroxylationof proline and lysine; phosphorylation of the hydroxyl groups of serylor threonyl residues; methylation of the alpha-amino groups of lysine,arginine and histidine; acetylation of the N-terminal amine; methylationof main chain amide residues or substitution with N-methyl amino acids;or amidation of C-terminal carboxyl groups.

A residue, e.g., an amino acid, of a polypeptide of the invention canalso be replaced by an amino acid (or peptidomimetic residue) of theopposite chirality. Thus, any amino acid naturally occurring in theL-configuration (which can also be referred to as the R or S, dependingupon the structure of the chemical entity) can be replaced with theamino acid of the same chemical structural type or a peptidomimetic, butof the opposite chirality, referred to as the D-amino acid, but also canbe referred to as the R- or S-form.

The invention also provides methods for modifying the polypeptides ofthe invention by either natural processes, such as post-translationalprocessing (e.g., phosphorylation, acylation, etc), or by chemicalmodification techniques, and the resulting modified polypeptides.Modifications can occur anywhere in the polypeptide, including thepeptide backbone, the amino acid side-chains and the amino or carboxyltermini. It will be appreciated that the same type of modification maybe present in the same or varying degrees at several sites in a givenpolypeptide. Also a given polypeptide may have many types ofmodifications. Modifications include acetylation, acylation,ADP-ribosylation, amidation, covalent attachment of flavin, covalentattachment of a heme moiety, covalent attachment of a nucleotide ornucleotide derivative, covalent attachment of a lipid or lipidderivative, covalent attachment of a phosphatidylinositol, cross-linkingcyclization, disulfide bond formation, demethylation, formation ofcovalent cross-links, formation of cysteine, formation of pyroglutamate,formylation, gamma-carboxylation, glycosylation, GPI anchor formation,hydroxylation, iodination, methylation, myristolyation, oxidation,pegylation, proteolytic processing, phosphorylation, prenylation,racemization, selenoylation, sulfation, and transfer-RNA mediatedaddition of amino acids to protein such as arginylation. See, e.g.,Creighton, T. E., Proteins—Structure and Molecular Properties 2nd Ed.,W.H. Freeman and Company, New York (1993); Posttranslational CovalentModification of Proteins, B. C. Johnson, Ed., Academic Press, New York,pp. 1-12 (1983).

Solid-phase chemical peptide synthesis methods can also be used tosynthesize the polypeptide or fragments of the invention. Such methodhave been known in the art since the early 1960's (Merrifield, R. B., J.Am. Chem. Soc., 85:2149-2154, 1963) (See also Stewart, J. M. and Young,J. D., Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co.,Rockford, Ill., pp. 11-12)) and have recently been employed incommercially available laboratory peptide design and synthesis kits(Cambridge Research Biochemicals). Such commercially availablelaboratory kits have generally utilized the teachings of H. M. Geysen etal, Proc. Natl. Acad. Sci., USA, 81:3998 (1984) and provide forsynthesizing peptides upon the tips of a multitude of “rods” or “pins”all of which are connected to a single plate. When such a system isutilized, a plate of rods or pins is inverted and inserted into a secondplate of corresponding wells or reservoirs, which contain solutions forattaching or anchoring an appropriate amino acid to the pin's or rod'stips. By repeating such a process step, i.e., inverting and insertingthe rod's and pin's tips into appropriate solutions, amino acids arebuilt into desired peptides. In addition, a number of available FMOCpeptide synthesis systems are available. For example, assembly of apolypeptide or fragment can be carried out on a solid support using anApplied Biosystems, Inc. Model 431A™ automated peptide synthesizer. Suchequipment provides ready access to the peptides of the invention, eitherby direct synthesis or by synthesis of a series of fragments that can becoupled using other known techniques.

The invention includes isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention withand without signal. The polypeptide comprising a signal sequence of theinvention can be an isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase of the invention or anotherisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase or another enzyme or other polypeptide.

The invention includes immobilized isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases andanti-isomerase, e.g., anti-racemase, e.g., anti-amino acid racemase,anti-alanine racemase, and/or anti-epimerase antibodies and fragmentsthereof. The invention provides methods for inhibiting isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseactivity, e.g., using dominant negative mutants or anti-isomerase, e.g.,anti-racemase, e.g., anti-amino acid racemase, anti-alanine racemase,and/or anti-epimerase antibodies of the invention. The inventionincludes heterocomplexes, e.g., fusion proteins, heterodimers, etc.,comprising the isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases of the invention.

Polypeptides of the invention can have an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase activityunder various conditions, e.g., extremes in pH and/or temperature,oxidizing agents, and the like. The invention provides methods leadingto alternative isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase preparations with different catalyticefficiencies and stabilities, e.g., towards temperature, oxidizingagents and changing wash conditions. In one aspect, isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasevariants can be produced using techniques of site-directed mutagenesisand/or random mutagenesis. In one aspect, directed evolution can be usedto produce a great variety of isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase variants withalternative specificities and stability.

The proteins of the invention are also useful as research reagents toidentify isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase modulators, e.g., activators or inhibitors ofisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase activity. Briefly, test samples (compounds, broths,extracts, and the like) are added to isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase assays todetermine their ability to inhibit substrate cleavage. Inhibitorsidentified in this way can be used in industry and research to reduce orprevent undesired proteolysis. Inhibitors can be combined to increasethe spectrum of activity.

The enzymes of the invention are also useful as research reagents todigest proteins or in protein sequencing. For example, the isomerases,e.g., racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases may be used to break polypeptides into smaller fragments forsequencing using, e.g. an automated sequencer.

The invention also provides methods of discovering new isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases using the nucleic acids, polypeptides and antibodies of theinvention. In one aspect, phagemid libraries are screened forexpression-based discovery of isomerases, e.g., racemases, e.g., aminoacid racemases, alanine racemases, and/or epimerases. In another aspect,lambda phage libraries are screened for expression-based discovery ofisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases. Screening of the phage or phagemidlibraries can allow the detection of toxic clones; improved access tosubstrate; reduced need for engineering a host, by-passing the potentialfor any bias resulting from mass excision of the library; and, fastergrowth at low clone densities. Screening of phage or phagemid librariescan be in liquid phase or in solid phase. In one aspect, the inventionprovides screening in liquid phase. This gives a greater flexibility inassay conditions; additional substrate flexibility; higher sensitivityfor weak clones; and ease of automation over solid phase screening.

The invention provides screening methods using the proteins and nucleicacids of the invention and robotic automation to enable the execution ofmany thousands of biocatalytic reactions and screening assays in a shortperiod of time, e.g., per day, as well as ensuring a high level ofaccuracy and reproducibility (see discussion of arrays, below). As aresult, a library of derivative compounds can be produced in a matter ofweeks. For further teachings on modification of molecules, includingsmall molecules, see PCT/US94/09174.

Another aspect of the invention is an isolated or purified polypeptidecomprising the sequence of one of the invention and sequencessubstantially identical thereto, or fragments comprising at least about5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive aminoacids thereof. As discussed above, such polypeptides may be obtained byinserting a nucleic acid encoding the polypeptide into a vector suchthat the coding sequence is operably linked to a sequence capable ofdriving the expression of the encoded polypeptide in a suitable hostcell. For example, the expression vector may comprise a promoter, aribosome binding site for translation initiation and a transcriptionterminator. The vector may also include appropriate sequences foramplifying expression.

Another aspect of the invention is polypeptides or fragments thereofwhich have at least about 50%, at least about 55%, at least about 60%,at least about 65%, at least about 70%, at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 95%,or more than about 95% homology to one of the polypeptides of theinvention and sequences substantially identical thereto, or a fragmentcomprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150consecutive amino acids thereof. Homology may be determined using any ofthe programs described above which aligns the polypeptides or fragmentsbeing compared and determines the extent of amino acid identity orsimilarity between them. It will be appreciated that amino acid“homology” includes conservative amino acid substitutions such as thosedescribed above.

The polypeptides or fragments having homology to one of the polypeptidesof the invention, or a fragment comprising at least about 5, 10, 15, 20,25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acids thereof maybe obtained by isolating the nucleic acids encoding them using thetechniques described above.

Alternatively, the homologous polypeptides or fragments may be obtainedthrough biochemical enrichment or purification procedures. The sequenceof potentially homologous polypeptides or fragments may be determined bygel electrophoresis and/or microsequencing. The sequence of theprospective homologous polypeptide or fragment can be compared to one ofthe polypeptides of the invention, or a fragment comprising at leastabout 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutiveamino acids thereof using any of the programs described above.

Another aspect of the invention is an assay for identifying fragments orvariants of The invention, which retain the enzymatic function of thepolypeptides of the invention. For example the fragments or variants ofsaid polypeptides, may be used to catalyze biochemical reactions, whichindicate that the fragment or variant retains the enzymatic activity ofthe polypeptides of the invention.

The assay for determining if fragments of variants retain the enzymaticactivity of the polypeptides of the invention includes the steps of:contacting the polypeptide fragment or variant with a substrate moleculeunder conditions which allow the polypeptide fragment or variant tofunction and detecting either a decrease in the level of substrate or anincrease in the level of the specific reaction product of the reactionbetween the polypeptide and substrate.

The present invention exploits the unique catalytic properties ofenzymes. Whereas the use of biocatalysts (i.e., purified or crudeenzymes, non-living or living cells) in chemical transformationsnormally requires the identification of a particular biocatalyst thatreacts with a specific starting compound, the present invention usesselected biocatalysts and reaction conditions that are specific forfunctional groups that are present in many starting compounds, such assmall molecules. Each biocatalyst is specific for one functional group,or several related functional groups and can react with many startingcompounds containing this functional group.

The biocatalytic reactions produce a population of derivatives from asingle starting compound. These derivatives can be subjected to anotherround of biocatalytic reactions to produce a second population ofderivative compounds. Thousands of variations of the original smallmolecule or compound can be produced with each iteration of biocatalyticderivatization.

Enzymes react at specific sites of a starting compound without affectingthe rest of the molecule, a process which is very difficult to achieveusing traditional chemical methods. This high degree of biocatalyticspecificity provides the means to identify a single active compoundwithin the library. The library is characterized by the series ofbiocatalytic reactions used to produce it, a so called “biosynthetichistory”. Screening the library for biological activities and tracingthe biosynthetic history identifies the specific reaction sequenceproducing the active compound. The reaction sequence is repeated and thestructure of the synthesized compound determined. This mode ofidentification, unlike other synthesis and screening approaches, doesnot require immobilization technologies and compounds can be synthesizedand tested free in solution using virtually any type of screening assay.It is important to note, that the high degree of specificity of enzymereactions on functional groups allows for the “tracking” of specificenzymatic reactions that make up the biocatalytically produced library.

Many of the procedural steps are performed using robotic automationenabling the execution of many thousands of biocatalytic reactions andscreening assays per day as well as ensuring a high level of accuracyand reproducibility. As a result, a library of derivative compounds canbe produced in a matter of weeks which would take years to produce usingcurrent chemical methods.

In a particular aspect, the invention provides a method for modifyingsmall molecules, comprising contacting a polypeptide encoded by apolynucleotide described herein or enzymatically active fragmentsthereof with a small molecule to produce a modified small molecule. Alibrary of modified small molecules is tested to determine if a modifiedsmall molecule is present within the library which exhibits a desiredactivity. A specific biocatalytic reaction which produces the modifiedsmall molecule of desired activity is identified by systematicallyeliminating each of the biocatalytic reactions used to produce a portionof the library and then testing the small molecules produced in theportion of the library for the presence or absence of the modified smallmolecule with the desired activity. The specific biocatalytic reactionswhich produce the modified small molecule of desired activity is in oneaspect (optionally) repeated. The biocatalytic reactions are conductedwith a group of biocatalysts that react with distinct structuralmoieties found within the structure of a small molecule, eachbiocatalyst is specific for one structural moiety or a group of relatedstructural moieties; and each biocatalyst reacts with many differentsmall molecules which contain the distinct structural moiety.

Isomerase Signal Sequences, Prepro and Catalytic Domains

The invention provides isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase signal sequences (e.g.,signal peptides (SPs)), prepro domains and catalytic domains (CDs). TheSPs, prepro domains and/or CDs of the invention can be isolated,synthetic or recombinant peptides or can be part of a fusion protein,e.g., as a heterologous domain in a chimeric protein. The inventionprovides nucleic acids encoding these catalytic domains (CDs), preprodomains and signal sequences (SPs, e.g., a peptide having a sequencecomprising/consisting of amino terminal residues of a polypeptide of theinvention). In one aspect, the invention provides a signal sequencecomprising a peptide comprising/consisting of a sequence as set forth inresidues 1 to 12, 1 to 13, 1 to 14, 1 to 15, 1 to 16, 1 to 17, 1 to 18,1 to 19, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 26,1 to 27, 1 to 28, 1 to 28, 1 to 30, 1 to 31, 1 to 32, 1 to 33, 1 to 34,1 to 35, 1 to 36, 1 to 37, 1 to 38, 1 to 39, 1 to 40, 1 to 41, 1 to 42,1 to 43, 1 to 44, 1 to 45, 1 to 46, 1 to 47, 1 to 48, 1 to 49 or 1 to50, of a polypeptide of the invention.

The isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase signal sequences (SPs) and/or preprosequences of the invention can be isolated peptides, or, sequencesjoined to another isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase or a non-isomerase, e.g.,non-racemase, e.g., non-amino acid racemase, non-alanine racemase,and/or non-epimerase polypeptide, e.g., as a fusion (chimeric) protein.In one aspect, the invention provides polypeptides comprising isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase signal sequences of the invention. In one aspect, polypeptidescomprising isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase signal sequences SPs and/or prepro of theinvention comprise sequences heterologous to an isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseof the invention (e.g., a fusion protein comprising an SP and/or preproof the invention and sequences from another isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase or anon-isomerase, e.g., non-racemase, e.g., non-amino acid racemase,non-alanine racemase, and/or non-epimerase polypeptide protein). In oneaspect, the invention provides isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase of the invention withheterologous SPs and/or prepro sequences, e.g., sequences with a yeastsignal sequence. An isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase of the invention cancomprise a heterologous SP and/or prepro in a vector, e.g., a pPICseries vector (Invitrogen, Carlsbad, Calif.).

In one aspect, SPs and/or prepro sequences of the invention areidentified following identification of novel isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerasepolypeptides. The pathways by which proteins are sorted and transportedto their proper cellular location are often referred to as proteintargeting pathways. One of the most important elements in all of thesetargeting systems is a short amino acid sequence at the amino terminusof a newly synthesized polypeptide called the signal sequence. Thissignal sequence directs a protein to its appropriate location in thecell and is removed during transport or when the protein reaches itsfinal destination. Most lysosomal, membrane, or secreted proteins havean amino-terminal signal sequence that marks them for translocation intothe lumen of the endoplasmic reticulum. More than 100 signal sequencesfor proteins in this group have been determined. The signal sequencescan vary in length from between about 10 to 50 amino acid residues.Various methods of recognition of signal sequences are known to those ofskill in the art. For example, in one aspect, novel isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasesignal peptides are identified by a method referred to as SignalP.SignalP uses a combined neural network which recognizes both signalpeptides and their cleavage sites; see, e.g., Nielsen (1997)“Identification of prokaryotic and eukaryotic signal peptides andprediction of their cleavage sites.” Protein Engineering 10:1-6.

It should be understood that in some aspects isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases of the invention may not have SPs and/or prepro sequences, or“domains.” In one aspect, the invention provides the isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases of the invention lacking all or part of an SP and/or a preprodomain. In one aspect, the invention provides a nucleic acid sequenceencoding a signal sequence (SP) and/or prepro from one isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseoperably linked to a nucleic acid sequence of a different isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase or, in one aspect (optionally), a signal sequence (SPs) and/orprepro domain from a non-isomerase, e.g., non-racemase, e.g., non-aminoacid racemase, non-alanine racemase, and/or non-epimerase protein may bedesired.

The invention also provides isolated, synthetic or recombinantpolypeptides comprising signal sequences (SPs), prepro domain and/orcatalytic domains (CDs) of the invention and heterologous sequences. Theheterologous sequences are sequences not naturally associated (e.g., toan isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase) with an SP, prepro domain and/or CD. Thesequence to which the SP, prepro domain and/or CD are not naturallyassociated can be on the SP's, prepro domain and/or CD's amino terminalend, carboxy terminal end, and/or on both ends of the SP and/or CD. Inone aspect, the invention provides an isolated, synthetic or recombinantpolypeptide comprising (or consisting of) a polypeptide comprising asignal sequence (SP), prepro domain and/or catalytic domain (CD) of theinvention with the proviso that it is not associated with any sequenceto which it is naturally associated (e.g., an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase sequence).Similarly in one aspect, the invention provides isolated, synthetic orrecombinant nucleic acids encoding these polypeptides. Thus, in oneaspect, the isolated, synthetic or recombinant nucleic acid of theinvention comprises coding sequence for a signal sequence (SP), preprodomain and/or catalytic domain (CD) of the invention and a heterologoussequence (i.e., a sequence not naturally associated with the a signalsequence (SP), prepro domain and/or catalytic domain (CD) of theinvention). The heterologous sequence can be on the 3′ terminal end, 5′terminal end, and/or on both ends of the SP, prepro domain and/or CDcoding sequence.

Hybrid (Chimeric) Isomerases and Peptide Libraries

In one aspect, the invention provides hybrid isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases and fusion proteins, including peptide libraries, comprisingsequences of the invention. The peptide libraries of the invention canbe used to isolate peptide modulators (e.g., activators or inhibitors)of targets, such as isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase substrates, receptors,enzymes. The peptide libraries of the invention can be used to identifyformal binding partners of targets, such as ligands, e.g., cytokines,hormones and the like. In one aspect, the invention provides chimericproteins comprising a signal sequence (SP), prepro domain and/orcatalytic domain (CD) of the invention or a combination thereof and aheterologous sequence (see above).

In one aspect, the fusion proteins of the invention (e.g., the peptidemoiety) are conformationally stabilized (relative to linear peptides) toallow a higher binding affinity for targets. The invention providesfusions of isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases of the invention and otherpeptides, including known and random peptides. They can be fused in sucha manner that the structure of the isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases is notsignificantly perturbed and the peptide is metabolically or structurallyconformationally stabilized. This allows the creation of a peptidelibrary that is easily monitored both for its presence within cells andits quantity.

Amino acid sequence variants of the invention can be characterized by apredetermined nature of the variation, a feature that sets them apartfrom a naturally occurring form, e.g., an allelic or interspeciesvariation of an isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase sequence. In one aspect, the variantsof the invention exhibit the same qualitative biological activity as thenaturally occurring analogue. Alternatively, the variants can beselected for having modified characteristics. In one aspect, while thesite or region for introducing an amino acid sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase variants screened for the optimalcombination of desired activity. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, as discussed herein for example, M13 primer mutagenesis and PCRmutagenesis. Screening of the mutants can be done using, e.g., assays ofisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase activity. In alternative aspects, amino acidsubstitutions can be single residues; insertions can be on the order offrom about 1 to 20 amino acids, although considerably larger insertionscan be done. Deletions can range from about 1 to about 20, 30, 40, 50,60, 70 residues or more. To obtain a final derivative with the optimalproperties, substitutions, deletions, insertions or any combinationthereof may be used. Generally, these changes are done on a few aminoacids to minimize the alteration of the molecule. However, largerchanges may be tolerated in certain circumstances.

The invention provides isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases where the structure ofthe polypeptide backbone, the secondary or the tertiary structure, e.g.,an alpha-helical or beta-sheet structure, has been modified. In oneaspect, the charge or hydrophobicity has been modified. In one aspect,the bulk of a side chain has been modified. Substantial changes infunction or immunological identity are made by selecting substitutionsthat are less conservative. For example, substitutions can be made whichmore significantly affect: the structure of the polypeptide backbone inthe area of the alteration, for example a alpha-helical or a beta-sheetstructure; a charge or a hydrophobic site of the molecule, which can beat an active site; or a side chain. The invention provides substitutionsin polypeptide of the invention where (a) a hydrophilic residues, e.g.seryl or threonyl, is substituted for (or by) a hydrophobic residue,e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g. lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g. glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.phenylalanine, is substituted for (or by) one not having a side chain,e.g. glycine. The variants can exhibit the same qualitative biologicalactivity (i.e. isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase activity) although variants can beselected to modify the characteristics of the isomerases, e.g.,racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases as needed.

In one aspect, isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases of the invention comprise epitopesor purification tags, signal sequences or other fusion sequences, etc.In one aspect, the isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention can befused to a random peptide to form a fusion polypeptide. By “fused” or“operably linked” herein is meant that the random peptide and theisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase are linked together, in such a manner as to minimizethe disruption to the stability of the structure, e.g., it retainsisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase activity. The fusion polypeptide (or fusionpolynucleotide encoding the fusion polypeptide) can comprise furthercomponents as well, including multiple peptides at multiple loops.

In one aspect, the peptides and nucleic acids encoding them arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. “Randomized” means that each nucleic acid and peptide consistsof essentially random nucleotides and amino acids, respectively. In oneaspect, the nucleic acids which give rise to the peptides can bechemically synthesized, and thus may incorporate any nucleotide at anyposition. Thus, when the nucleic acids are expressed to form peptides,any amino acid residue may be incorporated at any position. Thesynthetic process can be designed to generate randomized nucleic acids,to allow the formation of all or most of the possible combinations overthe length of the nucleic acid, thus forming a library of randomizednucleic acids. The library can provide a sufficiently structurallydiverse population of randomized expression products to affect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Thus, the inventionprovides an interaction library large enough so that at least one of itsmembers will have a structure that gives it affinity for some molecule,protein, or other factor.

Isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases are multidomain enzymes that consist in oneaspect (optionally) of a signal peptide, a catalytic domain, a linkerand/or another catalytic domain.

The invention provides a means for generating chimeric polypeptideswhich may encode biologically active hybrid polypeptides (e.g., hybridisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases). In one aspect, the originalpolynucleotides encode biologically active polypeptides. The method ofthe invention produces new hybrid polypeptides by utilizing cellularprocesses which integrate the sequence of the original polynucleotidessuch that the resulting hybrid polynucleotide encodes a polypeptidedemonstrating activities derived from the original biologically activepolypeptides. For example, the original polynucleotides may encode aparticular enzyme from different microorganisms. An enzyme encoded by afirst polynucleotide from one organism or variant may, for example,function effectively under a particular environmental condition, e.g.high salinity. An enzyme encoded by a second polynucleotide from adifferent organism or variant may function effectively under a differentenvironmental condition, such as extremely high temperatures. A hybridpolynucleotide containing sequences from the first and second originalpolynucleotides may encode an enzyme which exhibits characteristics ofboth enzymes encoded by the original polynucleotides. Thus, the enzymeencoded by the hybrid polynucleotide may function effectively underenvironmental conditions shared by each of the enzymes encoded by thefirst and second polynucleotides, e.g., high salinity and extremetemperatures.

A hybrid polypeptide resulting from the method of the invention mayexhibit specialized enzyme activity not displayed in the originalenzymes. For example, following recombination and/or reductivereassortment of polynucleotides encoding isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimeraseactivities, the resulting hybrid polypeptide encoded by a hybridpolynucleotide can be screened for specialized isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimeraseactivities obtained from each of the original enzymes, i.e. the type ofbond on which the isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase acts and the temperature at which theisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase functions. Thus, for example, the isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerasemay be screened to ascertain those chemical functionalities whichdistinguish the hybrid isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase from the originalisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases, for example, differences in activity atvarious temperatures, pH or salt concentration.

Sources of the original polynucleotides may be isolated from individualorganisms (“isolates”), collections of organisms that have been grown indefined media (“enrichment cultures”), or, uncultivated organisms(“environmental samples”). The use of a culture-independent approach toderive polynucleotides encoding novel bioactivities from environmentalsamples is most preferable since it allows one to access untappedresources of biodiversity.

“Environmental libraries” are generated from environmental samples andrepresent the collective genomes of naturally occurring organismsarchived in cloning vectors that can be propagated in suitableprokaryotic hosts. Because the cloned DNA is initially extracteddirectly from environmental samples, the libraries are not limited tothe small fraction of prokaryotes that can be grown in pure culture.Additionally, a normalization of the environmental DNA present in thesesamples could allow more equal representation of the DNA from all of thespecies present in the original sample. This can dramatically increasethe efficiency of finding interesting genes from minor constituents ofthe sample which may be under-represented by several orders of magnitudecompared to the dominant species.

For example, gene libraries generated from one or more uncultivatedmicroorganisms are screened for an activity of interest. Potentialpathways encoding bioactive molecules of interest are first captured inprokaryotic cells in the form of gene expression libraries.Polynucleotides encoding activities of interest are isolated from suchlibraries and introduced into a host cell. The host cell is grown underconditions which promote recombination and/or reductive reassortmentcreating potentially active biomolecules with novel or enhancedactivities.

Additionally, subcloning may be performed to further isolate sequencesof interest. In subcloning, a portion of DNA is amplified, digested,generally by restriction enzymes, to cut out the desired sequence, thedesired sequence is ligated into a recipient vector and is amplified. Ateach step in subcloning, the portion is examined for the activity ofinterest, in order to ensure that DNA that encodes the structuralprotein has not been excluded. The insert may be purified at any step ofthe subcloning, for example, by gel electrophoresis prior to ligationinto a vector or where cells containing the recipient vector and cellsnot containing the recipient vector are placed on selective mediacontaining, for example, an antibiotic, which will kill the cells notcontaining the recipient vector. Specific methods of subcloning cDNAinserts into vectors are well-known in the art (Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory Press (1989)). In another aspect, the enzymes of theinvention are subclones. Such subclones may differ from the parent cloneby, for example, length, a mutation, a tag or a label.

The microorganisms from which the polynucleotide may be prepared includeprokaryotic microorganisms, such as Eubacteria and Archaebacteria andlower eukaryotic microorganisms such as fungi, some algae and protozoa.Polynucleotides may be isolated from environmental samples in which casethe nucleic acid may be recovered without culturing of an organism orrecovered from one or more cultured organisms. In one aspect, suchmicroorganisms may be extremophiles, such as hyperthermophiles,psychrophiles, psychrotrophs, halophiles, barophiles and acidophiles.Polynucleotides encoding enzymes isolated from extremophilicmicroorganisms can be used. Such enzymes may function at temperaturesabove 100° C. in terrestrial hot springs and deep sea thermal vents, attemperatures below 0° C. in arctic waters, in the saturated saltenvironment of the Dead Sea, at pH values around 0 in coal deposits andgeothermal sulfur-rich springs, or at pH values greater than 11 insewage sludge. For example, several esterases and lipases cloned andexpressed from extremophilic organisms show high activity throughout awide range of temperatures and pHs.

Polynucleotides selected and isolated as hereinabove described areintroduced into a suitable host cell. A suitable host cell is any cellwhich is capable of promoting recombination and/or reductivereassortment. The selected polynucleotides are preferably already in avector which includes appropriate control sequences. The host cell canbe a higher eukaryotic cell, such as a mammalian cell, or a lowereukaryotic cell, such as a yeast cell, or preferably, the host cell canbe a prokaryotic cell, such as a bacterial cell. Introduction of theconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE-Dextran mediated transfection, or electroporation(Davis et al., 1986).

As representative examples of appropriate hosts, there may be mentioned:bacterial cells, such as E. coli, Streptomyces, Salmonella typhimurium;fungal cells, such as yeast; insect cells such as Drosophila S2 andSpodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma;adenoviruses; and plant cells. The selection of an appropriate host isdeemed to be within the scope of those skilled in the art from theteachings herein.

With particular references to various mammalian cell culture systemsthat can be employed to express recombinant protein, examples ofmammalian expression systems include the COS-7 lines of monkey kidneyfibroblasts, described in “SV40-transformed simian cells support thereplication of early SV40 mutants” (Gluzman, 1981) and other cell linescapable of expressing a compatible vector, for example, the C127, 3T3,CHO, HeLa and BHK cell lines. Mammalian expression vectors will comprisean origin of replication, a suitable promoter and enhancer and also anynecessary ribosome binding sites, polyadenylation site, splice donor andacceptor sites, transcriptional termination sequences and 5′ flankingnontranscribed sequences. DNA sequences derived from the SV40 splice andpolyadenylation sites may be used to provide the required nontranscribedgenetic elements.

In another aspect, it is envisioned the method of the present inventioncan be used to generate novel polynucleotides encoding biochemicalpathways from one or more operons or gene clusters or portions thereof.For example, bacteria and many eukaryotes have a coordinated mechanismfor regulating genes whose products are involved in related processes.The genes are clustered, in structures referred to as “gene clusters,”on a single chromosome and are transcribed together under the control ofa single regulatory sequence, including a single promoter whichinitiates transcription of the entire cluster. Thus, a gene cluster is agroup of adjacent genes that are either identical or related, usually asto their function. An example of a biochemical pathway encoded by geneclusters are polyketides.

Gene cluster DNA can be isolated from different organisms and ligatedinto vectors, particularly vectors containing expression regulatorysequences which can control and regulate the production of a detectableprotein or protein-related array activity from the ligated geneclusters. Use of vectors which have an exceptionally large capacity forexogenous DNA introduction are particularly appropriate for use withsuch gene clusters and are described by way of example herein to includethe f-factor (or fertility factor) of E. coli. This f-factor of E. coliis a plasmid which affects high-frequency transfer of itself duringconjugation and is ideal to achieve and stably propagate large DNAfragments, such as gene clusters from mixed microbial samples. Oneaspect of the invention is to use cloning vectors, referred to as“fosmids” or bacterial artificial chromosome (BAC) vectors. These arederived from E. coli f-factor which is able to stably integrate largesegments of genomic DNA. When integrated with DNA from a mixeduncultured environmental sample, this makes it possible to achieve largegenomic fragments in the form of a stable “environmental DNA library.”Another type of vector for use in the present invention is a cosmidvector. Cosmid vectors were originally designed to clone and propagatelarge segments of genomic DNA. Cloning into cosmid vectors is describedin detail in Sambrook et al., Molecular Cloning: A Laboratory Manual,2nd Ed., Cold Spring Harbor Laboratory Press (1989). Once ligated intoan appropriate vector, two or more vectors containing differentpolyketide synthase gene clusters can be introduced into a suitable hostcell. Regions of partial sequence homology shared by the gene clusterswill promote processes which result in sequence reorganization resultingin a hybrid gene cluster. The novel hybrid gene cluster can then bescreened for enhanced activities not found in the original geneclusters.

Therefore, in a one aspect, the invention relates to a method forproducing a biologically active hybrid polypeptide and screening such apolypeptide for enhanced activity by:

-   -   1) introducing at least a first polynucleotide in operable        linkage and a second polynucleotide in operable linkage, the at        least first polynucleotide and second polynucleotide sharing at        least one region of partial sequence homology, into a suitable        host cell;    -   2) growing the host cell under conditions which promote sequence        reorganization resulting in a hybrid polynucleotide in operable        linkage;    -   3) expressing a hybrid polypeptide encoded by the hybrid        polynucleotide;    -   4) screening the hybrid polypeptide under conditions which        promote identification of enhanced biological activity; and    -   5) isolating the a polynucleotide encoding the hybrid        polypeptide.

Methods for screening for various enzyme activities are known to thoseof skill in the art and are discussed throughout the presentspecification. Such methods may be employed when isolating thepolypeptides and polynucleotides of the invention.

Screening Methodologies and “on-Line” Monitoring Devices

In practicing the methods of the invention, a variety of apparatus andmethodologies can be used to in conjunction with the polypeptides andnucleic acids of the invention, e.g., to screen polypeptides forisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase activity, to screen compounds as potential modulators,e.g., activators or inhibitors, of an isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase activity, forantibodies that bind to a polypeptide of the invention, for nucleicacids that hybridize to a nucleic acid of the invention, to screen forcells expressing a polypeptide of the invention and the like. Inaddition to the array formats described in detail below for screeningsamples, alternative formats can also be used to practice the methods ofthe invention. Such formats include, for example, mass spectrometers,chromatographs, e.g., high-throughput HPLC and other forms of liquidchromatography, and smaller formats, such as 1536-well plates, 384-wellplates and so on. High throughput screening apparatus can be adapted andused to practice the methods of the invention, see, e.g., U.S. PatentApplication No. 20020001809.

Capillary Arrays

Nucleic acids or polypeptides of the invention can be immobilized to orapplied to an array. Arrays can be used to screen for or monitorlibraries of compositions (e.g., small molecules, antibodies, nucleicacids, etc.) for their ability to bind to or modulate the activity of anucleic acid or a polypeptide of the invention. Capillary arrays, suchas the GIGAMATRIX™, Diversa Corporation, San Diego, Calif.; and arraysdescribed in, e.g., U.S. Patent Application No. 20020080350 A1; WO0231203 A; WO 0244336 A, provide an alternative apparatus for holdingand screening samples. In one aspect, the capillary array includes aplurality of capillaries formed into an array of adjacent capillaries,wherein each capillary comprises at least one wall defining a lumen forretaining a sample. The lumen may be cylindrical, square, hexagonal orany other geometric shape so long as the walls form a lumen forretention of a liquid or sample. The capillaries of the capillary arraycan be held together in close proximity to form a planar structure. Thecapillaries can be bound together, by being fused (e.g., where thecapillaries are made of glass), glued, bonded, or clamped side-by-side.Additionally, the capillary array can include interstitial materialdisposed between adjacent capillaries in the array, thereby forming asolid planar device containing a plurality of through-holes.

A capillary array can be formed of any number of individual capillaries,for example, a range from 100 to 4,000,000 capillaries. Further, acapillary array having about 100,000 or more individual capillaries canbe formed into the standard size and shape of a Microtiter® plate forfitment into standard laboratory equipment. The lumens are filledmanually or automatically using either capillary action ormicroinjection using a thin needle. Samples of interest may subsequentlybe removed from individual capillaries for further analysis orcharacterization. For example, a thin, needle-like probe is positionedin fluid communication with a selected capillary to either add orwithdraw material from the lumen.

In a single-pot screening assay, the assay components are mixed yieldinga solution of interest, prior to insertion into the capillary array. Thelumen is filled by capillary action when at least a portion of the arrayis immersed into a solution of interest. Chemical or biologicalreactions and/or activity in each capillary are monitored for detectableevents. A detectable event is often referred to as a “hit”, which canusually be distinguished from “non-hit” producing capillaries by opticaldetection. Thus, capillary arrays allow for massively parallel detectionof “hits”.

In a multi-pot screening assay, a polypeptide or nucleic acid, e.g., aligand, can be introduced into a first component, which is introducedinto at least a portion of a capillary of a capillary array. An airbubble can then be introduced into the capillary behind the firstcomponent. A second component can then be introduced into the capillary,wherein the second component is separated from the first component bythe air bubble. The first and second components can then be mixed byapplying hydrostatic pressure to both sides of the capillary array tocollapse the bubble. The capillary array is then monitored for adetectable event resulting from reaction or non-reaction of the twocomponents.

In a binding screening assay, a sample of interest can be introduced asa first liquid labeled with a detectable particle into a capillary of acapillary array, wherein the lumen of the capillary is coated with abinding material for binding the detectable particle to the lumen. Thefirst liquid may then be removed from the capillary tube, wherein thebound detectable particle is maintained within the capillary, and asecond liquid may be introduced into the capillary tube. The capillaryis then monitored for a detectable event resulting from reaction ornon-reaction of the particle with the second liquid.

Arrays, or “Biochips”

Nucleic acids and/or polypeptides of the invention can be immobilized toor applied to an array, e.g., a “biochip”. Arrays can be used to screenfor or monitor libraries of compositions (e.g., small molecules,antibodies, nucleic acids, etc.) for their ability to bind to ormodulate the activity of a nucleic acid or a polypeptide of theinvention. For example, in one aspect of the invention, a monitoredparameter is transcript expression of an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase gene. Oneor more, or, all the transcripts of a cell can be measured byhybridization of a sample comprising transcripts of the cell, or,nucleic acids representative of or complementary to transcripts of acell, by hybridization to immobilized nucleic acids on an array, or“biochip.” By using an “array” of nucleic acids on a microchip, some orall of the transcripts of a cell can be simultaneously quantified.Alternatively, arrays comprising genomic nucleic acid can also be usedto determine the genotype of a newly engineered strain made by themethods of the invention. Polypeptide arrays” can also be used tosimultaneously quantify a plurality of proteins. The present inventioncan be practiced with any known “array,” also referred to as a“microarray” or “nucleic acid array” or “polypeptide array” or “antibodyarray” or “biochip,” or variation thereof. Arrays are generically aplurality of “spots” or “target elements,” each target elementcomprising a defined amount of one or more biological molecules, e.g.,oligonucleotides, immobilized onto a defined area of a substrate surfacefor specific binding to a sample molecule, e.g., mRNA transcripts.

The terms “array” or “microarray” or “biochip” or “chip” as used hereinis a plurality of target elements, each target element comprising adefined amount of one or more polypeptides (including antibodies) ornucleic acids immobilized onto a defined area of a substrate surface, asdiscussed in further detail, below.

In practicing the methods of the invention, any known array and/ormethod of making and using arrays can be incorporated in whole or inpart, or variations thereof, as described, for example, in U.S. Pat.Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695;6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174;5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522;5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g.,WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g.,Johnston (1998) Curr. Biol. 8:R171-R174; Schummer (1997) Biotechniques23:1087-1092; Kern (1997) Biotechniques 23:120-124; Solinas-Toldo (1997)Genes, Chromosomes & Cancer 20:399-407; Bowtell (1999) Nature GeneticsSupp. 21:25-32. See also published U.S. patent applications Nos.20010018642; 20010019827; 20010016322; 20010014449; 20010014448;20010012537; 20010008765.

Antibodies and Antibody-Based Screening Methods

The invention provides isolated, synthetic or recombinant antibodiesthat specifically bind to an isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase of the invention. Theseantibodies can be used to isolate, identify or quantify an isomerase,e.g., racemase, e.g., amino acid racemase, alanine racemase, and/orepimerase of the invention or related polypeptides. These antibodies canbe used to isolate other polypeptides within the scope the invention orother related isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases. The antibodies can be designed tobind to an active site of an isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase. Thus, the inventionprovides methods of inhibiting isomerases, e.g., racemases, e.g., aminoacid racemases, alanine racemases, and/or epimerases using theantibodies of the invention (see discussion above regarding applicationsfor anti-isomerase, e.g., anti-racemase, e.g., anti-amino acid racemase,anti-alanine racemase, and/or anti-epimerase compositions of theinvention).

The invention provides fragments of the enzymes of the invention,including immunogenic fragments of a polypeptide of the invention. Theinvention provides compositions comprising a polypeptide or peptide ofthe invention and adjuvants or carriers and the like.

The antibodies can be used in immunoprecipitation, staining,immunoaffinity columns, and the like. If desired, nucleic acid sequencesencoding for specific antigens can be generated by immunization followedby isolation of polypeptide or nucleic acid, amplification or cloningand immobilization of polypeptide onto an array of the invention.Alternatively, the methods of the invention can be used to modify thestructure of an antibody produced by a cell to be modified, e.g., anantibody's affinity can be increased or decreased. Furthermore, theability to make or modify antibodies can be a phenotype engineered intoa cell by the methods of the invention.

The term “antibody” includes a peptide or polypeptide derived from,modeled after or substantially encoded by an immunoglobulin gene orimmunoglobulin genes, or fragments thereof, capable of specificallybinding an antigen or epitope, see, e.g. Fundamental Immunology, ThirdEdition, W. E. Paul, ed., Raven Press, N.Y. (1993); Wilson (1994) J.Immunol. Methods 175:267-273; Yarmush (1992) J. Biochem. Biophys.Methods 25:85-97. The term antibody includes antigen-binding portions,i.e., “antigen binding sites,” (e.g., fragments, subsequences,complementarity determining regions (CDRs)) that retain capacity to bindantigen, including (i) a Fab fragment, a monovalent fragment consistingof the VL, VH, CL and CH1 domains; (ii) a F(ab′)2 fragment, a bivalentfragment comprising two Fab fragments linked by a disulfide bridge atthe hinge region; (iii) a Fd fragment consisting of the VH and CH1domains; (iv) a Fv fragment consisting of the VL and VH domains of asingle arm of an antibody, (v) a dAb fragment (Ward et al., (1989)Nature 341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR). Single chain antibodies arealso included by reference in the term “antibody.”

Methods of immunization, producing and isolating antibodies (polyclonaland monoclonal) are known to those of skill in the art and described inthe scientific and patent literature, see, e.g., Coligan, CURRENTPROTOCOLS IN IMMUNOLOGY, Wiley/Greene, N.Y. (1991); Stites (eds.) BASICAND CLINICAL IMMUNOLOGY (7th ed.) Lange Medical Publications, Los Altos,Calif. (“Stites”); Goding, MONOCLONAL ANTIBODIES: PRINCIPLES ANDPRACTICE (2d ed.) Academic Press, New York, N.Y. (1986); Kohler (1975)Nature 256:495; Harlow (1988) ANTIBODIES, A LABORATORY MANUAL, ColdSpring Harbor Publications, New York. Antibodies also can be generatedin vitro, e.g., using recombinant antibody binding site expressing phagedisplay libraries, in addition to the traditional in vivo methods usinganimals. See, e.g., Hoogenboom (1997) Trends Biotechnol. 15:62-70; Katz(1997) Annu. Rev. Biophys. Biomol. Struct. 26:27-45.

The polypeptides of The invention or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof, may also be used to generate antibodies which bind specificallyto the polypeptides or fragments. The resulting antibodies may be usedin immunoaffinity chromatography procedures to isolate or purify thepolypeptide or to determine whether the polypeptide is present in abiological sample. In such procedures, a protein preparation, such as anextract, or a biological sample is contacted with an antibody capable ofspecifically binding to one of the polypeptides of The invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof.

In immunoaffinity procedures, the antibody is attached to a solidsupport, such as a bead or other column matrix. The protein preparationis placed in contact with the antibody under conditions in which theantibody specifically binds to one of the polypeptides of The invention,or fragment thereof. After a wash to remove non-specifically boundproteins, the specifically bound polypeptides are eluted.

The ability of proteins in a biological sample to bind to the antibodymay be determined using any of a variety of procedures familiar to thoseskilled in the art. For example, binding may be determined by labelingthe antibody with a detectable label such as a fluorescent agent, anenzymatic label, or a radioisotope. Alternatively, binding of theantibody to the sample may be detected using a secondary antibody havingsuch a detectable label thereon. Particular assays include ELISA assays,sandwich assays, radioimmunoassays and Western Blots.

Polyclonal antibodies generated against the polypeptides of Theinvention, or fragments comprising at least 5, 10, 15, 20, 25, 30, 35,40, 50, 75, 100, or 150 or more consecutive amino acids thereof can beobtained by direct injection of the polypeptides into an animal or byadministering the polypeptides to an animal, for example, a nonhuman.The antibody so obtained will then bind the polypeptide itself. In thismanner, even a sequence encoding only a fragment of the polypeptide canbe used to generate antibodies which may bind to the whole nativepolypeptide. Such antibodies can then be used to isolate the polypeptidefrom cells expressing that polypeptide.

For preparation of monoclonal antibodies, any technique which providesantibodies produced by continuous cell line cultures can be used.Examples include the hybridoma technique (Kohler and Milstein, Nature,256:495-497, 1975), the trioma technique, the human B-cell hybridomatechnique (Kozbor et al., Immunology Today 4:72, 1983) and theEBV-hybridoma technique (Cole, et al., 1985, in Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce single chain antibodies tothe polypeptides of The invention, or fragments comprising at least 5,10, 15, 20, 25, 30, 35, 40, 50, 75, 100, or 150 consecutive amino acidsthereof. Alternatively, transgenic mice may be used to express humanizedantibodies to these polypeptides or fragments thereof.

Antibodies generated against the polypeptides of The invention, orfragments comprising at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, or 150 consecutive amino acids thereof may be used in screening forsimilar polypeptides from other organisms and samples. In suchtechniques, polypeptides from the organism are contacted with theantibody and those polypeptides which specifically bind the antibody aredetected. Any of the procedures described above may be used to detectantibody binding. One such screening assay is described in “Methods forMeasuring Cellulase Activities”, Methods in Enzymology, Vol 160, pp.87-116.

Kits

The invention provides kits comprising the compositions, e.g., nucleicacids, expression cassettes, vectors, cells, transgenic seeds or plantsor plant parts, polypeptides (e.g., isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases) and/orantibodies of the invention. The kits also can contain instructionalmaterial teaching the methodologies and industrial, research, medical,pharmaceutical, food and feed and food and feed supplement processingand other applications and processes of the invention, as describedherein.

Whole Cell Engineering and Measuring Metabolic Parameters

The methods of the invention provide whole cell evolution, or whole cellengineering, of a cell to develop a new cell strain having a newphenotype, e.g., a new or modified isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase activity, bymodifying the genetic composition of the cell. The genetic compositioncan be modified by addition to the cell of a nucleic acid of theinvention, e.g., a coding sequence for an enzyme of the invention. See,e.g., WO0229032; WO0196551.

To detect the new phenotype, at least one metabolic parameter of amodified cell is monitored in the cell in a “real time” or “on-line”time frame. In one aspect, a plurality of cells, such as a cell culture,is monitored in “real time” or “on-line.” In one aspect, a plurality ofmetabolic parameters is monitored in “real time” or “on-line.” Metabolicparameters can be monitored using the isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases of theinvention.

Metabolic flux analysis (MFA) is based on a known biochemistryframework. A linearly independent metabolic matrix is constructed basedon the law of mass conservation and on the pseudo-steady statehypothesis (PSSH) on the intracellular metabolites. In practicing themethods of the invention, metabolic networks are established, includingthe:

-   -   identity of all pathway substrates, products and intermediary        metabolites    -   identity of all the chemical reactions interconverting the        pathway metabolites, the stoichiometry of the pathway reactions,    -   identity of all the enzymes catalyzing the reactions, the enzyme        reaction kinetics,    -   the regulatory interactions between pathway components, e.g.        allosteric interactions, enzyme-enzyme interactions etc,    -   intracellular compartmentalization of enzymes or any other        supramolecular organization of the enzymes, and,    -   the presence of any concentration gradients of metabolites,        enzymes or effector molecules or diffusion barriers to their        movement.

Once the metabolic network for a given strain is built, mathematicpresentation by matrix notion can be introduced to estimate theintracellular metabolic fluxes if the on-line metabolome data isavailable. Metabolic phenotype relies on the changes of the wholemetabolic network within a cell. Metabolic phenotype relies on thechange of pathway utilization with respect to environmental conditions,genetic regulation, developmental state and the genotype, etc. In oneaspect of the methods of the invention, after the on-line MFAcalculation, the dynamic behavior of the cells, their phenotype andother properties are analyzed by investigating the pathway utilization.For example, if the glucose supply is increased and the oxygen decreasedduring the yeast fermentation, the utilization of respiratory pathwayswill be reduced and/or stopped, and the utilization of the fermentativepathways will dominate. Control of physiological state of cell cultureswill become possible after the pathway analysis. The methods of theinvention can help determine how to manipulate the fermentation bydetermining how to change the substrate supply, temperature, use ofinducers, etc. to control the physiological state of cells to move alongdesirable direction. In practicing the methods of the invention, the MFAresults can also be compared with transcriptome and proteome data todesign experiments and protocols for metabolic engineering or geneshuffling, etc.

In practicing the methods of the invention, any modified or newphenotype can be conferred and detected, including new or improvedcharacteristics in the cell. Any aspect of metabolism or growth can bemonitored.

Monitoring Expression of an mRNA Transcript

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of an mRNA transcript (e.g., anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase message) or generating new (e.g., isomerase, e.g.,racemase, e.g., amino acid racemase, alanine racemase, and/or epimerase)transcripts in a cell. This increased or decreased expression can betraced by testing for the presence of an isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase of theinvention or by isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase activity assays. mRNA transcripts, ormessages, also can be detected and quantified by any method known in theart, including, e.g., Northern blots, quantitative amplificationreactions, hybridization to arrays, and the like. Quantitativeamplification reactions include, e.g., quantitative PCR, including,e.g., quantitative reverse transcription polymerase chain reaction, orRT-PCR; quantitative real time RT-PCR, or “real-time kinetic RT-PCR”(see, e.g., Kreuzer (2001) Br. J. Haematol. 114:313-318; Xia (2001)Transplantation 72:907-914).

In one aspect of the invention, the engineered phenotype is generated byknocking out expression of a homologous gene. The gene's coding sequenceor one or more transcriptional control elements can be knocked out,e.g., promoters or enhancers. Thus, the expression of a transcript canbe completely ablated or only decreased.

In one aspect of the invention, the engineered phenotype comprisesincreasing the expression of a homologous gene. This can be effected byknocking out of a negative control element, including a transcriptionalregulatory element acting in cis- or trans-, or, mutagenizing a positivecontrol element. One or more, or, all the transcripts of a cell can bemeasured by hybridization of a sample comprising transcripts of thecell, or, nucleic acids representative of or complementary totranscripts of a cell, by hybridization to immobilized nucleic acids onan array.

Monitoring Expression of a Polypeptides, Peptides and Amino Acids

In one aspect of the invention, the engineered phenotype comprisesincreasing or decreasing the expression of a polypeptide (e.g., anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase) or generating new polypeptides in a cell. Thisincreased or decreased expression can be traced by determining theamount of isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase present or by isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase activityassays. Polypeptides, peptides and amino acids also can be detected andquantified by any method known in the art, including, e.g., nuclearmagnetic resonance (NMR), spectrophotometry, radiography (proteinradiolabeling), electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, various immunological methods,e.g. immunoprecipitation, immunodiffusion, immuno-electrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immuno-fluorescent assays, gel electrophoresis (e.g., SDS-PAGE),staining with antibodies, fluorescent activated cell sorter (FACS),pyrolysis mass spectrometry, Fourier-Transform Infrared Spectrometry,Raman spectrometry, GC-MS, and LC-Electrospray andcap-LC-tandem-electrospray mass spectrometries, and the like. Novelbioactivities can also be screened using methods, or variations thereof,described in U.S. Pat. No. 6,057,103. Furthermore, as discussed below indetail, one or more, or, all the polypeptides of a cell can be measuredusing a protein array.

Industrial, Energy, Pharmaceutical, Medical, Food Processing and OtherApplications

Polypeptides of the invention can be used in or to make any industrial,agricultural, food and feed and food and feed supplement or food or feedadditive, pharmaceutical, medical, research (laboratory) or othercompositions or process. The invention provides industrial processesusing enzymes of the invention, e.g., in the pharmaceutical or nutrient(diet) supplement industry, the energy industry (e.g., to make “clean”biofuels), in the food and feed industries, e.g., in methods for makingfood and feed products and food and feed additives. In one aspect, theinvention provides processes using enzymes of the invention in themedical industry, e.g., to make pharmaceuticals, pharmaceuticalintermediates, or dietary aids or supplements, or food supplements andadditives. In addition, the invention provides methods for using theenzymes of the invention in biofuel production, including, e.g., abioalcohol such as bioethanol, biomethanol, biobutanol or biopropanol,thus comprising a “clean” fuel production.

The isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase enzymes of the invention can be highlyselective catalysts. They can catalyze reactions with exquisite stereo-,regio- and chemo-selectivities that are unparalleled in conventionalsynthetic chemistry. Moreover, enzymes are remarkably versatile. Theisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase enzymes of the invention can be tailored to function inorganic solvents, operate at extreme pHs (for example, high pHs and lowpHs) extreme temperatures (for example, high temperatures and lowtemperatures), extreme salinity levels (for example, high salinity andlow salinity) and catalyze reactions with compounds that arestructurally unrelated to their natural, physiological substrates.

In one aspect, the isomerases, e.g., racemases, e.g., amino acidracemases (e.g., resolving a D- and/or an L-isomer from a racemicmixture), alanine racemases, and/or epimerases of the invention are usedin processes in the manufacture of medicaments (pharmaceuticals, drugs,and/or their precursors or intermediates), pesticides and/or theirprecursors or intermediates thereof. For example, D-serine can be usefulfor binding to NMDA brain receptors to promote neuromodulation, orD-aspartate can be useful for hormonal regulation in endocrine tissues.

In one aspect, the invention uses an amino acid racemase of thisinvention to make (resolve) a D-amino acid by using a racemic and/or anL-amino acid source, the process comprising enzyme reaction on aspecific amino acid of interest. In one aspect, the invention uses anamino acid racemase of this invention to make a D-amino acid (from aracemate and/or an L-amino acid source) resistant to proteolysis. In oneaspect, the invention uses an amino acid racemase of this invention tomake (resolve) proteins comprising D-amino acids (from racemates and/orL-amino acid sources) to generate a new or enhanced antibiotic orimmunogenic property.

In one embodiment, the isomerase, e.g., racemase, e.g., amino acidracemase, alanine racemase, and/or epimerase enzymes of the inventionare used as targets for examining protein turnover in response to apathological or biological process, e.g. liver damage/disease ormyocardial infarction. In one exemplary transamination reaction using anenzyme of the invention, an alpha-amino group is transferred to analpha-carbon atom of an alpha-ketoglutarate generating the correspondingalpha-keto acid analog of the amino acid.

Detergent, Disinfectant and Cleaning Compositions

The invention provides cleaning compositions, e.g., detergent,disinfectant or cleanser (cleaning or cleansing) compositions, e.g. forcleaning fabrics, dishwashing, laundry, oral cleaning, denture cleaning,and contact lenses, comprising one or more polypeptides (e.g.,isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases) of the invention, and methods of makingand using these compositions. The invention incorporates all methods ofmaking and using detergent, disinfectant or cleanser compositions, see,e.g., U.S. Pat. Nos. 6,413,928; 6,399,561; 6,365,561; 6,380,147. Thedetergent, disinfectant or cleanser compositions can be a one and twopart aqueous composition, a non-aqueous liquid composition, a castsolid, a granular form, a particulate form, a compressed tablet, a geland/or a paste and a slurry form. The isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases of theinvention can also be used as a detergent, disinfectant or cleanseradditive product in a solid or a liquid form. Such additive products areintended to supplement or boost the performance of conventionaldetergent compositions and can be added at any stage of the cleaningprocess.

The actual active enzyme content depends upon the method of manufactureof a detergent, disinfectant or cleanser composition and is notcritical, assuming the detergent solution has the desired enzymaticactivity. In one aspect, the amount of isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase present in thefinal solution ranges from about 0.001 mg to 0.5 mg per gram of thedetergent composition. The particular enzyme chosen for use in theprocess and products of this invention depends upon the conditions offinal utility, including the physical product form, use pH, usetemperature, and soil types to be degraded or altered. The enzyme can bechosen to provide optimum activity and stability for any given set ofutility conditions. In one aspect, the isomerases, e.g., racemases,e.g., amino acid racemases, alanine racemases, and/or epimerases of thepresent invention are active in the pH ranges of from about 4 to about12 and in the temperature range of from about 20° C. to about 95° C. Thedetergents of the invention can comprise cationic, semi-polar nonionicor zwitterionic surfactants; or, mixtures thereof.

Isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention can be formulated intopowdered and liquid detergents, disinfectants or cleansers having pHbetween 4.0 and 12.0 at levels of about 0.01 to about 5% (preferably0.1% to 0.5%) by weight. These detergent, disinfectant or cleansercompositions can also include other isomerases, e.g., racemases, e.g.,amino acid racemases, alanine racemases, and/or epimerases and/or otherenzymes such as xylanases, cellulases, lipases, esterases, proteases, orendoglycosidases, endo-beta.-1,4-glucanases, beta-glucanases,endo-beta-1,3(4)-glucanases, cutinases, peroxidases, catalases,laccases, amylases, glucoamylases, pectinases, oxidoreductases,reductases, oxidases, transferases, transaminases, amino transferases,dehydrogenases, phenoloxidases, ligninases, pullulanases, arabinanases,hemicellulases, mannanases, xyloglucanases, pectin acetyl esterases,rhamnogalacturonan acetyl esterases, polygalacturonases,rhamnogalacturonases, galactanases, pectin lyases, pectinmethylesterases, cellobiohydrolases and/or transglutaminases. Thesedetergent, disinfectant or cleanser compositions can also include dyes,colorants, odorants, bleaches, buffers, builders, enzyme “enhancingagents” (see, e.g., U.S. Patent application no. 20030096394) andstabilizers.

In one aspect, the invention provides a method for cleaning or washingan object comprising contacting the object with a polypeptide of theinvention under conditions sufficient for cleaning or washing. Anisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase of the invention may be included as a detergent,disinfectant or cleanser additive. A fabric softener composition cancomprise an isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase of the invention.

Treating Foods and Food Processing

The isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention have numerous applicationsin food processing industry. For example, in one aspect, the isomerases,e.g., racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases of the invention are used to improve the extraction of oilfrom oil-rich plant material, e.g., oil-rich seeds, for example, soybeanoil from soybeans, olive oil from olives, rapeseed oil from rapeseedand/or sunflower oil from sunflower seeds. In another aspect, theisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention can be used for separationof components of plant cell materials.

The isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention can be used in thepreparation of fruit or vegetable juices, syrups, extracts and the liketo increase yield. The isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention can beused in the enzymatic treatment of various plant cell wall-derivedmaterials or waste materials, e.g. from cereals, grains, wine or juiceproduction, or agricultural residues such as vegetable hulls, beanhulls, sugar beet pulp, olive pulp, potato pulp, and the like. Theisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention can be used to modify theconsistency and appearance of processed fruit or vegetables. Theisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention can be used to treat plantmaterial to facilitate processing of plant material, including foods,facilitate purification or extraction of plant components.

In one aspect, isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases of the invention are used in bakingapplications, e.g., cookies, breads and crackers. In one aspect,isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention are used as additives indough processing. In another aspect of the invention, the isomerases,e.g., racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases of the invention can also be used in any food or beveragetreatment or food or beverage production process. In another aspect ofthe invention, the isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention can beincluded in any food or beverage composition.

Feeds and Food or Feed or Food Additives

The invention provides methods for treating feeds, foods, food or feedadditives, food or feed supplements, or dietary aids, using isomerases,e.g., racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases of the invention, animals including mammals (e.g., humans),birds, fish and the like. The invention provides feeds, foods, food orfeed additives, food or feed supplements, or dietary aids comprisingisomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention. In one aspect, treatingfeeds, foods, food or feed additives, food or feed supplements, ordietary aids using isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention canhelp in the availability of nutrients, e.g., starch, protein, sugars,and the like, in the feeds, foods, food or feed additives, food or feedsupplements, or dietary aids.

The feeds, foods, food or feed additives, food or feed supplements, ordietary aids of the invention may be a granulated, pelletized orparticulate form, which may be coated or uncoated. Alternatively, thefeeds, foods, food or feed additives, food or feed supplements, ordietary aids of the invention may be a stabilized liquid composition.This may be an aqueous or oil-based slurry. See, e.g., U.S. Pat. No.6,245,546.

In another aspect, isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention can besupplied by expressing the enzymes directly in transgenic feed crops(as, e.g., transgenic plants, seeds and the like), such as grains,cereals, corn, soy bean, rape seed, lupin and the like. As discussedabove, the invention provides transgenic plants, plant parts and plantcells comprising a nucleic acid sequence encoding a polypeptide of theinvention. In one aspect, the nucleic acid is expressed such that theisomerase, e.g., racemase, e.g., amino acid racemase, alanine racemase,and/or epimerase of the invention is produced in recoverable quantities.The isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases can be recovered from any plant or plantpart. Alternatively, the plant or plant part containing the recombinantpolypeptide can be used as such for improving the quality of a food orfeed, e.g., improving nutritional value, palatability, and rheologicalproperties, or to destroy an antinutritive factor.

A coating can be applied to the invention enzyme granules, pellets,particles for many different purposes, such as to add a flavor ornutrition supplement, to delay release nutrients and enzymes in gastricconditions, and the like. Or, the coating may be applied to achieve afunctional goal, for example, whenever it is desirable to slow releaseof the enzyme from the matrix particles or to control the conditionsunder which the enzyme will be released. The composition of the coatingmaterial can be such that it is selectively broken down by an agent towhich it is susceptible (such as heat, acid or base, enzymes or otherchemicals). Alternatively, two or more coatings susceptible to differentsuch breakdown agents may be consecutively applied to the matrixparticles.

The invention is also directed towards a process for preparing anenzyme-releasing matrix. In accordance with the invention, the processcomprises providing discrete plural particles of a grain-based substratein a particle size suitable for use as an enzyme-releasing matrix,wherein the particles comprise an isomerase, e.g., racemase, e.g., aminoacid racemase, alanine racemase, and/or epimerase enzyme encoded by anamino acid sequence of the invention or at least 30 consecutive aminoacids thereof. Preferably, the process includes compacting orcompressing the particles of enzyme-releasing matrix into granules,which can be accomplished by pelletizing. The mold inhibitor andcohesiveness agent, when used, can be added at any suitable time, andcan be mixed with the grain-based substrate in the desired proportionsprior to pelletizing of the grain-based substrate. Moisture content inthe pellet mill feed can be in the ranges set forth above with respectto the moisture content in the finished product, and can be about14-15%. In one aspect, moisture is added to the feedstock in the form ofan aqueous preparation of the enzyme to bring the feedstock to thismoisture content. The temperature in the pellet mill can be brought toabout 82° C. with steam. The pellet mill may be operated under anyconditions that impart sufficient work to the feedstock to providepellets. The pelleting process itself is a cost-effective process forremoving water from the enzyme-containing composition.

In one aspect, the pellet mill is operated with a ⅛ in. by 2 inch die at100 lb./min pressure at 82° C. to provide pellets, which then arecrumbled in a pellet mill crumbler to provide discrete plural particleshaving a particle size capable of passing through an 8 mesh screen butbeing retained on a 20 mesh screen.

The thermostable isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention can beused in the pellets of the invention. They can have high optimumtemperatures and high heat resistance such that an enzyme reaction at atemperature not hitherto carried out can be achieved. The gene encodingthe isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase according to the present invention (e.g. asset forth in any of the sequences in the invention) can be used inpreparation of isomerases, e.g., racemases, e.g., amino acid racemases,alanine racemases, and/or epimerases (e.g. using GSSM as describedherein) having characteristics different from those of the isomerases,e.g., racemases, e.g., amino acid racemases, alanine racemases, and/orepimerases of the invention (in terms of optimum pH, optimumtemperature, heat resistance, stability to solvents, specific activity,affinity to substrate, secretion ability, translation rate,transcription control and the like).

Waste Treatment

The isomerases, e.g., racemases, e.g., amino acid racemases, alanineracemases, and/or epimerases of the invention can be used in a varietyof other industrial applications, e.g., in waste treatment. For example,in one aspect, the invention provides a solid waste digestion processusing isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase of the invention. The methods can comprisereducing the mass and volume of substantially untreated solid waste.Solid waste can be treated with an enzymatic digestive process in thepresence of an enzymatic solution (including isomerase, e.g., racemase,e.g., amino acid racemase, alanine racemase, and/or epimerase of theinvention) at a controlled temperature. This results in a reactionwithout appreciable bacterial fermentation from added microorganisms.The solid waste is converted into a liquefied waste and any residualsolid waste. The resulting liquefied waste can be separated from saidany residual solidified waste. See e.g., U.S. Pat. No. 5,709,796.

In another aspect of the invention, the isomerase, e.g., racemase, e.g.,amino acid racemase, alanine racemase, and/or epimerase of the inventioncan also be used in any waste treatment process. In another aspect ofthe invention, the isomerase, e.g., racemase, e.g., amino acid racemase,alanine racemase, and/or epimerase of the invention can be included inany waste treatment composition.

Oral Care Products

The invention provides oral care products comprising enzymes of thisinvention, such as trans isomerases, e.g., racemases, e.g., amino acidracemases, alanine racemases, and/or epimerases of the invention,including the enzyme mixtures or “cocktails” of the invention. Exemplaryoral care products include toothpastes, dental creams, gels or toothpowders, odontics, mouth washes, pre- or post brushing rinseformulations, chewing gums, lozenges, or candy. See, e.g., U.S. Pat. No.6,264,925.

Biomass Conversion and Biofuel Production

The invention provides methods and processes for biomass conversion orany organic material to a fuel, e.g., to a fuel, e.g. a biofuel, such asbioethanol, biomethanol, biopropanol and/or biobutanol and the like,using enzymes of the invention, including the enzyme mixtures or“cocktails” of the invention. Thus, the invention provides fuels, e.g.,biofuels, such as bioethanols, comprising a polypeptide of theinvention, including the enzyme mixtures or “cocktails” of theinvention, or a polypeptide encoded by a nucleic acid of the invention.In alternative aspects, the fuel is derived from a plant material, whichoptionally comprises potatoes, soybean (rapeseed), barley, rye, corn,oats, wheat, beets or sugar cane, and optionally the fuel comprises abioethanol or a gasoline-ethanol mix.

The invention provides methods for making a fuel comprising contacting abiomass composition or any organic material with a polypeptide of theinvention, or a polypeptide encoded by a nucleic acid of the invention,or any one of the mixtures or “cocktails” or products of manufacture ofthe invention. In alternative embodiments, the biomass compositioncomprises a plant, plant product or plant derivative, and the plant orplant product can comprise cane sugar plants or plant products, beets orsugarbeets, wheat, corn, soybeans, potato, rice or barley. In oneaspect, the fuel comprises a bioethanol or a gasoline-ethanol mix, or abiopropanol or a gasoline-propanol mix, or a biobutanol or agasoline-butanol mix, or a biomethanol or a gasoline-methanol mix, or abiodiesel or a gasoline-biodiesel mix, or any combination thereof.

The invention provides methods for making bioethanol, biobutanol,biomethanol and/or a biopropanol comprising contacting a biomasscomposition or any organic material with a polypeptide of the invention,or a polypeptide encoded by a nucleic acid of the invention, or any oneof the mixtures or “cocktails” or products of manufacture of theinvention. In alternative embodiments, the biomass composition comprisesa plant, plant product or plant derivative, and the plant or plantproduct can comprise cane sugar plants or plant products, beets orsugarbeets, wheat, corn, soybeans, potato, rice or barley. Inalternative embodiments, the organic material or biomass is derived froman agricultural crop (e.g., wheat, barley, potatoes, switchgrass, poplarwood), is a byproduct of a food or a feed production, is alignocellulosic waste product, or is a plant residue or a waste paper orwaste paper product, and optionally the plant residue comprise stems,leaves, hulls, husks, corn or corn cobs, corn stover, corn fiber, hay,straw (e.g. rice straw or wheat straw), sugarcane bagasse, sugar beetpulp, citrus pulp, and citrus peels, wood, wood thinnings, wood chips,wood pulp, pulp waste, wood waste, wood shavings and sawdust,construction and/or demolition wastes and debris (e.g. wood, woodshavings and sawdust), and optionally the paper waste comprisesdiscarded or used photocopy paper, computer printer paper, notebookpaper, notepad paper, typewriter paper, newspapers, magazines, cardboardand paper-based packaging materials, and recycled paper materials. Inaddition, urban wastes, e.g. the paper fraction of municipal solidwaste, municipal wood waste, and municipal green waste, can be used.

The invention provides compositions (including products of manufacture,enzyme ensembles, or “cocktails”) comprising a mixture (or “cocktail”)of isomerase, e.g., racemase, e.g., amino acid racemase, alanineracemase, and/or epimerase enzymes.

The invention provides cells and/or organisms expressing enzymes of theinvention (e.g., wherein the cells or organisms comprise as heterologousnucleic acids a sequence of this invention) for participation inchemical cycles involving natural biomass (e.g., plant) conversion.Alternatively, the polypeptide of the invention may be expressed in thebiomass plant material or feedstock itself.

The methods of the invention also include taking the converted biomass(e.g., lignocellulosic) material (processed by enzymes of the invention)and making it into a fuel (e.g. a biofuel such as a bioethanol,biobutanol, biomethanol, a biopropanol, or a biodiesel) by fermentationand/or by chemical synthesis. In one aspect, the produced sugars arefermented and/or the non-fermentable products are gasified.

The methods of the invention also include converting algae, virginvegetable oils, waste vegetable oils, animal fats and greases (e.g.tallow, lard, and yellow grease), or sewage, using enzymes of theinvention, and making it into a fuel (e.g. a bioalcohol, e.g., abioethanol, biomethanol, biobutanol or biopropanol, or biodiesel) byfermentation and/or by chemical synthesis or conversion.

The enzymes of the invention (including, for example, organisms, such asmicroorganisms, e.g., fungi, yeast or bacteria, and plants and plantcells and plant parts, e.g., seeds, making and in some aspects secretingrecombinant enzymes of the invention) can be used in orincluded/integrated at any stage of any organic matter/biomassconversion process, e.g., at any one step, several steps, or included inall of the steps, or all of the following methods of biomass conversionprocesses, or all of these biofuel alternatives:

-   -   Direct combustion: the burning of material by direct heat and is        the simplest biomass technology; can be very economical if a        biomass source is nearby.        -   1 Pyrolysis: is the thermal degradation of biomass by heat            in the absence of oxygen. In one aspect, biomass is heated            to a temperature between about 800 and 1400 degrees            Fahrenheit, but no oxygen is introduced to support            combustion resulting in the creation of gas, fuel oil and            charcoal.        -   2 Gasification: biomass can be used to produce methane            through heating or anaerobic digestion. Syngas, a mixture of            carbon monoxide and hydrogen, can be derived from biomass.    -   Landfill Gas: is generated by the decay (anaerobic digestion) of        buried garbage in landfills. When the organic waste decomposes,        it generates gas consisting of approximately 50% methane, the        major component of natural gas.    -   Anaerobic digestion: converts organic matter to a mixture of        methane, the major component of natural gas, and carbon dioxide.        In one aspect, biomass such as waterwaste (sewage), manure, or        food processing waste, is mixed with water and fed into a        digester tank without air.    -   Fermentation        -   Alcohol Fermentation: fuel alcohol is produced by converting            cellulosic mass and/or starch to sugar, fermenting the sugar            to alcohol, then separating the alcohol water mixture by            distillation. Feedstocks such as dedicated crops (e.g.,            wheat, barley, potatoes, switchgrass, poplar wood),            agricultural residues and wastes (e.g. rice straw, corn            stover, wheat straw, sugarcane bagasse, rice hulls, corn            fiber, sugar beet pulp, citrus pulp, and citrus peels),            forestry wastes (e.g. hardwood and softwood thinnings,            hardwood and softwood residues from timber operations, wood            shavings, and sawdust), urban wastes (e.g. paper fraction of            municipal solid waste, municipal wood waste, municipal green            waste), wood wastes (e.g. saw mill waste, pulp mill waste,            construction waste, demolition waste, wood shavings, and            sawdust), and waste paper or other materials containing            sugar, starch, and/or cellulose can be converted to sugars            and then to alcohol by fermentation with yeast.            Alternatively, materials containing sugars can be converted            directly to alcohol by fermentation.    -   Transesterification: An exemplary reaction for converting oil to        biodiesel is called transesterification. The transesterification        process reacts an alcohol (like methanol) with the triglyceride        oils contained in vegetable oils, animal fats, or recycled        greases, forming fatty acid alkyl esters (biodiesel) and        glycerin. The reaction requires heat and a strong base catalyst,        such as sodium hydroxide or potassium hydroxide.    -   Biodiesel: Biodiesel is a mixture of fatty acid alkyl esters        made from vegetable oils, animal fats or recycled greases.        Biodiesel can be used as a fuel for vehicles in its pure form,        but it is usually used as a petroleum diesel additive to reduce        levels of particulates, carbon monoxide, hydrocarbons and air        toxics from diesel-powered vehicles.    -   Hydrolysis: includes hydrolysis of a compound, e.g., a biomass,        such as a lignocellulosic material, catalyzed using an enzyme of        the instant invention.    -   Cogeneration: is the simultaneous production of more than one        form of energy using a single fuel and facility. In one aspect,        biomass cogeneration has more potential growth than biomass        generation alone because cogeneration produces both heat and        electricity.

Enzymes of the invention can also be used in glycerin refining. Theglycerin by-product contains unreacted catalyst and soaps that areneutralized with an acid. Water and alcohol are removed to produce 50%to 80% crude glycerin. The remaining contaminants include unreacted fatsand oils, which can be processes using the polypeptides of theinvention. In a large biodiesel plants of the invention, the glycerincan be further purified, e.g., to 99% or higher purity, for thepharmaceutical and cosmetic industries.

Biofuels as a Liquid or a Gas Gasoline

The invention provides biofuels and synthetic fuels in the form of agas, or gasoline, e.g., a syngas. In one aspect, methods of theinvention comprising use of enzymes of the invention for chemical cyclesfor natural biomass conversion, e.g., for the hydrolysis of a biomass tomake a biofuel, e.g., a bioethanol, biopropanol, bio-butanol or abiomethanol, or a synthetic fuel, in the form of a liquid or as a gas,such as a “syngas”.

For example, invention provides methods for making biofuel gases andsynthetic gas fuels (“syngas”) comprising a bioethanol, biopropanol,bio-butanol and/or a biomethanol made using a polypeptide of theinvention, or made using a method of the invention; and in one aspectthis biofuel gas of the invention is mixed with a natural gas (can alsobe produced from biomass), e.g., a hydrogen or a hydrocarbon-based gasfuel. In one aspect, the invention provides methods for processingbiomass to a synthetic fuel, e.g., a syngas, such as a syngas producedfrom a biomass by gasification. In one aspect, the invention providesmethods for making an ethanol, propanol, butanol and/or methanol gasfrom a sugar cane, e.g., a bagasse. In one aspect, this fuel, or gas, isused as motor fuel, e.g., an automotive, truck, airplane, boat, smallengine, etc. fuel. In one aspect, the invention provides methods formaking an ethanol, propanol, butanol and/or methanol from a plant, e.g.,corn, or a plant product, e.g., hay or straw (e.g., a rice straw or awheat straw, or any the dry stalk of any cereal plant), or anagricultural waste product.

In one aspect, the ethanol, propanol, butanol and/or methanol made usinga method of composition of the invention can be used as a fuel (e.g., agasoline) additive (e.g., an oxygenator) or in a direct use as a fuel.For example, a ethanol, propanol, butanol and/or methanol, including afuel, made by a method of the invention can be mixed with ethyl tertiarybutyl ether (ETBE), or an ETBE mixture such as ETBE containing 47%ethanol as a biofuel, or with MTBE (methyl tertiary-butyl ether). Inanother aspect, a ethanol, propanol, butanol and/or methanol, includinga fuel, made by a method of the invention can be mixed with:

IUPAC name Common name but-1-ene α-butylene cis-but-2-ene cis-β-butylenetrans-but-2-ene trans-β-butylene 2-methylpropene isobutylene

A butanol and/or ethanol made by a method of the invention (e.g., usingan enzyme of the invention) can be further processed using “A.B.E.”(Acetone, Butanol, Ethanol) fermentation; in one aspect, butanol beingthe only liquid product. In one aspect, this butanol and/or ethanol isburned “straight” in existing gasoline engines (without modification tothe engine or car), produces more energy and is less corrosive and lesswater soluble than ethanol, and can be distributed via existinginfrastructures.

In one aspect, one, several or all of these alcohols are made by aprocess of the invention using an enzyme of the invention, and theprocess can further comprise a biomass-to-liquid technology, e.g., agasification process to produce syngas followed by catalytic synthesis,or by a bioconversion of biomass to a mixed alcohol fuel.

The invention also provides processes comprising use of an enzyme of theinvention incorporating (or, incorporated into) “gas to liquid”, or GTL;or “coal to liquid”, or CTL; or “biomass to liquid” or BTL; or “oilsandsto liquid”, or OTL, processes; and in one aspect these processes of theinvention are used to make synthetic fuels. In one aspect, one of theseprocesses of the invention comprises making a biofuel (e.g., a synfuel)out of a biomass using, e.g., the so-called “Fischer Tropsch” process (acatalyzed chemical reaction in which carbon monoxide and hydrogen areconverted into liquid hydrocarbons of various forms; typical catalystsused are based on iron and cobalt; the principal purpose of this processis to produce a synthetic petroleum substitute for use as syntheticlubrication oil or as synthetic fuel). In one aspect, this syntheticbiofuel of the invention can contain oxygen and can be used as additivein high quality diesel and petrol.

In alternative aspects, the processes of the invention use variouspretreatments, which can be grouped into three categories: physical,chemical, and multiple (physical+chemical). Any chemicals can be used asa pretreatment agent, e.g., acids, alkalis, gases, cellulose solvents,alcohols, oxidizing agents and reducing agents. Among these chemicals,alkali is the most popular pretreatment agent because it is relativelyinexpensive and results in less cellulose degradation. The commonalkalis sodium hydroxide and lime also can be used as pretreatmentagents. Although sodium hydroxide increases biomass digestibilitysignificantly, it is difficult to recycle, is relatively expensive, andis dangerous to handle. In contrast, lime has many advantages: it issafe and very inexpensive, and can be recovered by carbonating washwater with carbon dioxide.

In one aspect, the invention provides a biofuel, e.g., a biogas,produced by the process of anaerobic digestion of organic material byanaerobes, wherein the process comprises use of an enzyme of theinvention or a method of the invention. This biofuel, e.g., a biogas,can be produced either from biodegradable waste materials or by the useof energy crops fed into anaerobic digesters to supplement gas yields.The solid output, digestate, can also be used as a biofuel.

The invention provides methods for making biologically produced oils,including crude oils, and gases that can be used in diesel engines,wherein the process comprises use of an enzyme of the invention or amethod of the invention. In one aspect, these methods can refinepetroleum, e.g., crude oils, into kerosene, petroleum, diesel and otherfractions.

The invention provides methods (using an enzyme of the invention or amethod of the invention) for making biologically produced oils from:

-   -   Straight vegetable oil (SVO).    -   Waste vegetable oil (WVO)—waste cooking oils and greases        produced in quantity mostly by commercial kitchens.    -   Biodiesel obtained from transesterification of animal fats and        vegetable oil, directly usable in petroleum diesel engines.    -   Biologically derived crude oil, together with biogas and carbon        solids via the thermal depolymerization of complex organic        materials including non oil based materials; for example, waste        products such as old tires, offal, wood and plastic.    -   Pyrolysis oil; which may be produced out of biomass, wood waste        etc. using heat only in the flash pyrolysis process (the oil may        have to be treated before using in conventional fuel systems or        internal combustion engines).    -   Wood, charcoal, and dried dung.

The invention will be further described with reference to the followingexamples; however, it is to be understood that the invention is notlimited to such examples.

EXAMPLES

The Examples in Part A describe the methodologies used for initialcharacterization of the candidate isomerase and epimerase nucleic acidsand the encoded polypeptides. Further characterization of the isomeraseand epimerase nucleic acids and polypeptides is described in Part B.

Part A Example 1 Effect of Leader Sequence on Racemase

Many of the racemases that were discovered had native signal/leadersequences. The signal sequences and corresponding cleavage sites wereidentified by SignalP 3.0 (at cbs.dtu.dk/services/SignalP/ on the WorldWide Web). It was observed that clones containing racemases with leadersequences tended to be more difficult to grow. The clones grew well withfresh transformations, however they did not grow well when they weresubcultured or inoculated from glycerol stocks. Samples were grown (or,at least, attempted) a minimum of two times.

The table below indicates several clones that contained their nativesignal sequences. These samples were in the PCR4-TOPO vector/E. coliTop10 host (Invitrogen, Carlsbad, Calif.). Growth conditions wereover-night in LB/kanamycin 50 μg/mL, 37° C. All of these samples weredifficult to grow. The consensus leader sequence for the samples shownbelow is: MHKKTLLATLIFGLLAGQAVA (SEQ ID NO:499). 19 of the clones havethis leader sequence exactly. 17 of the clones have a leader sequencethat differs by one amino acid: MHKKTLLATLILGLLAGQAVA (SEQ ID NO:500).Therefore, the consensus sequence for racemase leader sequences isMHKKTLLATLIXGLLAGQAVA (SEQ ID NO:501) where X is F or L.

TABLE 4 Leadered racemase clones Clone Clone Clone Clone SEQ ID NO: 170SEQ ID NO: 180 SEQ ID NO: 134 SEQ ID NO: 118 SEQ ID NO: 108 SEQ ID NO:182 SEQ ID NO: 146 SEQ ID NO: 194 SEQ ID NO: 172 SEQ ID NO: 184 SEQ IDNO: 112 SEQ ID NO: 154 SEQ ID NO: 136 SEQ ID NO: 140 SEQ ID NO: 114 SEQID NO: 156 SEQ ID NO: 110 SEQ ID NO: 142 SEQ ID NO: 148 SEQ ID NO: 196SEQ ID NO: 174 SEQ ID NO: 186 SEQ ID NO: 116 SEQ ID NO: 158 SEQ ID NO:138 SEQ ID NO: 144 SEQ ID NO: 150 SEQ ID NO: 120 SEQ ID NO: 176 SEQ IDNO: 188 SEQ ID NO: 192 SEQ ID NO: 160 SEQ ID NO: 178 SEQ ID NO: 190 SEQID NO: 152 SEQ ID NO: 162

The leadered racemase clone (Pseudomonas putida KT2440 BAR—that was notdifficult to grow. The leadered racemase clone (Pseudomonas putidaKT2440 BAR was in the pET30 vector (Novagen, Madison, Wis.)/E. coliexpression host BL21(DE3) (Novagen, Madison, Wis.). The leader sequencefor the Pseudomonas putida KT2440 BAR was determined to be:MPFRRTLLAASLALLITGQAPLYA (SEQ ID NO:502).

To further investigate the effect of the leader sequence on growth, someracemases were subcloned into expression vectors with and without thenative signal sequences. The samples are listed below (Table 5). Theleft-hand column indicates the leadered subclone version while themiddle column indicates the same gene subcloned without a leadersequence (for example, SEQ ID NO:412 is the leaderless version of SEQ IDNO:490). These samples were in the pSE420-cHis vector/E. coli MB2946host (Strych & Benedik, 2002, J. Bacteriology, 184:4321-5). The his-tagwas not expressed in these constructs. Growth conditions were over-nightin LB/carbenicillin 100 μg/mL, 37° C.

TABLE 5 Racemases that were subcloned with and without the native leadersequence Subclone Subclone w/ counterpart native leader w/out leaderComments SEQ ID NO: 490 SEQ ID NO: 412 leadered version difficult togrow SEQ ID NO: 492 SEQ ID NO: 400 no difficulties growing eitherversion SEQ ID NO: 494 SEQ ID NO: 408 leadered version difficult to growSEQ ID NO: 496 SEQ ID NO: 410 leadered version difficult to grow SEQ IDNO: 498 SEQ ID NO: 402 leadered version difficult to grow SEQ ID NO: 428SEQ ID NO: 404 leadered version does not reach OD600 = 0.5 within 8hours; could not induce SEQ ID NO: 430 SEQ ID NO: 406 leadered versiondoes not reach OD600 = 0.5 within 8 hours; could not induce

In general, the leadered racemase subclones were more difficult to growthan the non-leadered counterparts under the conditions described inPart A. SEQ ID NO:490, SEQ ID NO:494, SEQ ID NO:496 and SEQ ID NO:498were difficult to grow. SEQ ID NO:428 and SEQ ID NO:430 would growhowever they grew extremely slowly and did not reach an inducibleOD₆₀₀=0.5 within 8 hours. SEQ ID NO:492 was the only leadered racemasesubclone tested that was not difficult to grow.

In summary, leadered racemase candidates generally were harder to growthan the non-leadered counterparts under the conditions described above.The reason for the decrease in viability or robustness has not beenidentified. The cells could potentially be expelling the plasmids,thereby losing the antibiotic resistance over time. In order to maximizerobustness, the number of rounds of growth for racemases with leadersequences was minimized. This was done by storing the DNA and performingfresh transformations each time the constructs were used.

The host organisms, expression conditions, and post expression cellhandling can all affect whether there is detectable tryptophan racemaseactivity under the conditions of the assay in the presence of therespective leader sequences. Additionally, under optimized conditions,it is expected that all racemase candidates could have tryptophanracemase activity with or without leader sequences (native or artificialsuch as PelB).

Example 2 Improvement of SEQ ID NO:412 Solubility Using ARCTICEXPRESS™Hosts

The expression of SEQ ID NO:412 racemase was analyzed by SDS-PAGE. SEQID NO:412 expressed well and had high activity even though only aportion (<20%) of the protein was soluble. In order to improve solubleexpression, the racemase was moved into two ARCTICEXPRESS™ hosts(Stratagene, La Jolla, Calif.). The racemase was subcloned into thepET28b vector and the DNA was transformed into ArcticExpress™ (DE3) andArcticExpress™ (DE3)RIL and plated on LB kanamycin 50 μg/mL, gentamicin20 μg/mL, and LB kanamycin 50 μg/mL, gentamicin 20 μg/mL, streptomycin75 μg/mL, respectively. pET28b vector control DNA was also transformedinto each host. Samples were grown overnight at 30° C. Four colonieswere picked for each construct from each ArcticExpress™ host.

TABLE 6 Names of constructs in ArcticExpress ™(DE3) &ArcticExpress ™(DE3)RIL Name Description DE3-1 SEQ ID NO: 412 racemaseORF in pET28b/ArcticExpress ™(DE3) -colony #1 DE3-2 SEQ ID NO: 412racemase ORF in pET28b/ArcticExpress ™(DE3) -colony #2 DE3-3 SEQ ID NO:412 racemase ORF in pET28b/ArcticExpress ™(DE3) -colony #3 DE3-4 SEQ IDNO: 412 racemase ORF in pET28b/ArcticExpress ™(DE3) -colony #4 DE3-VpET28b/ArcticExpress ™(DE3) RIL-1 SEQ ID NO: 412 racemase ORF inpET28b/ArcticExpress ™(DE3)RIL -colony #1 RIL-2 SEQ ID NO: 412 racemaseORF in pET28b/ArcticExpress ™(DE3)RIL -colony #2 RIL-3 SEQ ID NO: 412racemase ORF in pET28b/ArcticExpress ™(DE3)RIL -colony #3 RIL-3 SEQ IDNO: 412 racemase ORF in pET28b/ArcticExpress ™(DE3)RIL -colony #4 RIL-VpET28b/ArcticExpress ™(DE3)RIL

Cultures were streaked onto fresh plates with the appropriateantibiotics, two days prior to performing a large scale growth. Sampleswere grown on LB plates with the appropriate antibiotics and incubatedovernight at 30° C. The next day, a single colony was picked from eachplate and used to inoculate 50 mL of LB with appropriate antibiotics.Samples were incubated overnight at 30° C. and 210 rpm. The next day,the culture was used to inoculate 500 mLs of LB with the appropriateantibiotics in a 2.8 L baffled flask to OD₆₀₀=0.05. The cultures weregrown at 30° C. at 210 rpm. When the OD₆₀₀ was between 0.4-0.8, theflasks were transferred to an 11° C. incubator and allowed to incubatefor 10 minutes prior to inducing with 1 mM IPTG Samples were inducedovernight at 11° C. at 210 rpm (with the exception of DE3-2 and DE3-4,which were induced at 16° C.).

The next morning the cultures were collected and centrifuged at 6,000rpm for 20 minutes, and the supernatant was discarded. The pellet wasresuspended in 20 mL of 50 mM sodium phosphate buffer (pH 7.5), 400μg/mL lysozyme, 26 U/mL DNase I. Cells were lysed using a microfluidizer(Microfluidics Corporation, Newton, Mass.) per the manufacturer'sinstructions; each sample was passed through the microfluidizer threetimes. 1 mL of lysate was set aside for gel analysis of the totalprotein fraction. The remainder of the lysate was centrifuged at 12,000rpm at 4° C. for 30 minutes. The supernatant was saved. Proteinconcentration was determined using the Bio-Rad Protein Assay (Bio-Rad,Hercules, Calif.). The soluble and whole cell fraction was then analyzedby SDS-PAGE using 4-20% Tris-glycine gels (Invitrogen, Carlsbad,Calif.).

TABLE 7 Soluble expression levels of racemase constructs inpET28b/ArcticExpress ™(DE3) and pET28b/ ArcticExpress ™(DE3)RIL NameSoluble Expression DE3-1 <75% DE3-2 <75% DE3-3 <75% DE3-4 <75% RIL-1<75% RIL-2 <50% RIL-3 <75% RIL-4 <50%

As shown above, the soluble expression of the racemase was improved inthe ArcticExpress™ (DE3) & ArcticExpress™ (DE3)RIL host.

Samples were tested for activity using a racemase assay (as described inExample 4). Racemases were loaded at 7.5, 0.75, 0.075 μg/mL totalprotein and incubated with 10 mM L-tryptophan and 10 μM PLP at pH 8 and37° C. At indicated timepoints, 50 μL of the reaction product was addedto 150 μL of ice cold acetonitrile. Samples were vortexed for 30 secondsand the supernatant was then diluted fifty-fold in methanol. Sampleswere then analyzed by LC/MS/MS (as described in Example 4) to monitorthe D-tryptophan formed and the residual L-tryptophan.

TABLE 8 Racemase activity of SEQ ID NO: 412 constructs inArcticExpress(DE3) and ArcticExpress(DE3)RIL μg/ml D-tryptophan formedName 0 hrs 1.75 hrs 3.75 hrs 19.75 hrs DE3-1 13.58 1010 1488 1278 DE3-24.24 1044 1130 1044 DE3-3 4 1018 1180 1166 DE3-4 5.46 1060 1070 1112RIL-1 14.2 1128 1178 1564 RIL-2 13.2 976 1078 1110 RIL-3 10.2 954 11221134 RIL-4 7.98 1008 1164 1134 7.5 μg/mL total protein

As shown above, all of the constructs were active in ArcticExpress(DE3)& ArcticExpress(DE3)RIL at a 7.5 μg/mL total protein load. All theconstructs were also active when the protein was loading at 0.75 and0.075 μg/mL total protein. The vector/host controls had little or noactivity compared to the racemase constructs.

In summary, the SEQ ID NO:412 racemase was active and soluble expressionwas improved in ArcticExpress™ (DE3) & ArcticExpress™ (DE3)RIL.

Example 3 Activity of Racemase PFAM Domain Subclones

Several sets of proprietary degenerate PCR primers were designed as partof a sequence-based discovery effort for the amplification of racemasesfrom mixed population environmental DNA libraries as described in U.S.Pat. No. 6,455,254. One set of proprietary degenerate PCR primersamplified the PFAM domain of the racemase exclusively. The racemaseswere amplified using a sequence-based discovery method (see U.S. Pat.No. 6,455,254). The PFAM domain is slightly smaller than the full-lengthracemase protein. As compared to the full length racemase, the PFAMdomain is missing about 30-40 amino acids from the N-terminus (mostlysignal peptide) and about 10-20 amino acids from the C-terminus

Several racemase PFAM domains were amplified using this method. ThreePFAM domains were selected for subcloning in order to determine if thePFAM domain was sufficient to detect racemase activity. The samples weresubcloned into the pSE420-cHis vector (his-tag not expressed) in E. coliMB2946 host cells (Strych & Benedik, 2002, J. Bacteriology, 184:4321-5).The subclones were SEQ ID NO:122, SEQ ID NO:440 and SEQ ID NO:462.

SEQ ID NO:122 was selected for activity testing. Flasks containing 50 mLLB, 100 μg/mL carbenicillin and 50 mM D-alanine were inoculated fromglycerol stocks and grown overnight at 37° C. with shaking. Thefollowing morning, flasks containing 400 mL LB, 100 μg/mL carbenicillinand 50 mM D-alanine were inoculated to OD₆₀₀=0.05. Cultures were grownat 37° C. with shaking and induced with 1 mM IPTG when OD₆₀₀=0.5-0.8.Cultures were induced overnight at 30° C.

Cell pellets were collected by centrifugation at 6000 rpm for 20minutes. Cell pellets were resuspended in 20 mL of 50 mM sodiumphosphate buffer pH 7.5 with 26 U/ml DNase I. Cell pellets were lysed ina microfluidizer (Microfluidics Corporation, Newton, Mass.) per themanufacturer's instructions. Samples were centrifuged at 12,000 rpm for30 minutes and the soluble fraction was collected. Protein concentrationwas determined by comparing the absorbance of SEQ ID NO:122 cell extractto known standards in the Bio-Rad Protein Assay reagent (Bio-Rad,Hercules, Calif.).

Samples were tested for activity using the following racemase assayconditions (also as described in Example 4). Racemases were loaded at 10mg/mL total protein and incubated with 10 mM L-tryptophan and 10 μM PLPat pH 8 and a temperature of 37° C. At indicated timepoints (0, 2, 4,and 24 hours), 50 μL of the reaction product was added to 150 μL ofice-cold acetonitrile. Samples were vortexed for 30 seconds and passedthrough a 0.2 μm filter and the filtrate was then diluted fifty-fold inmethanol. Samples were then analyzed by LC/MS/MS (as described inExample 4) to monitor the D-tryptophan formed (Table 9).

TABLE 9 Racemase activity of PFAM μg/ml D-tryptophan Name Description 0hr 2 hr 4 hr 24 hr E. coli MB2946 host Negative 0.26 0.18 0.22 0.22cells control Pseudomonas putida Positive control 0.23 58.97 88.35180.41 KT2440 BAR (leadered) SEQ ID NO: 122 Racemase 0.67 33.84 58.18106.69 PFAM No enzyme 0.18 0.21 0.21 0.18

Negative control—E. coli MB2946 host cells (Strych & Benedik, supra)

The leadered racemase clone (Pseudomonas putida KT2440 BAR—SEQ ID NO:122was active under the conditions described in Example 4. The resultsabove thereby demonstrate that a racemase PFAM domain could besufficient to detect racemase activity.

Example 4 Growth and Racemase Assay Procedures

Enzyme Preparation

Glycerol stocks were used to inoculate flasks containing 50 mLs of LBwith the appropriate antibiotic. The starter culture was grown overnightat 37° C. and 230 rpm. The OD_(600 nm) of starter culture was checked,and used to inoculate a 400 ml culture to an OD_(600 nm) of 0.05. Theculture was incubated at 37° C. and 230 rpm, and the OD_(600 nm) waschecked periodically. The cultures were induced, usually with 1 mM IPTG,when the OD_(600 nm) reached between 0.5-0.8. Induced cultures wereincubated overnight at 30° C. and 230 rpm. The culture was harvested bypelleting cells at 4000 rpm for 15 minutes. The supernatant was pouredoff, and either frozen for later use or the cells lysed.

The pellets were resuspended in 20 mls of 50 mM sodium phosphate buffer(pH 7.5) supplemented with 26 U/ml of DNase. Once the pellet wascompletely resuspended in the buffer, cells were lysed using amicrofluidizer (Microfluidics Corporation, Newton, Mass.) per themanufacturer's instructions. The clarified lysate was collected andcentrifuged at 11,000 rpm for 30 minutes. The supernatant was collectedin a clean tube and filtered through a 0.2 μm filter. 5 mls aliquots ofclarified lysate were placed in each vial and freeze-dried using thelyophilizer (Virtis Company, Gardinier, N.Y.) per the manufacturer'sinstructions. A 1 ml sample was retained for protein estimation usingthe Bio-Rad Protein Assay Reagent (Bio-Rad, Hercules, Calif.) andSDS-PAGE analysis. Once the lysate was lyophilized, the amount ofprotein per vial was calculated.

Enzymes were prepared for the activity assay by resuspending in 50 mMsodium phosphate (pH 7.5). The racemase assays were usually run withabout 10-20 mg/ml total protein.

Racemase Assay

10 mM L-tryptophan, 10 μM PLP, 50 mM sodium phosphate pH 8, 10 mg/mLracemase (total protein) prepared as described above (see Example4—Enzyme Preparation) were combined and incubated at 37° C. and 300 rpm.50 μL of the reaction product were transferred to 150 μL of ice coldacetonitrile at timepoints (generally 0, 2, 4, and 24 hours) and thesamples were vortexed for 30 seconds. The samples were centrifuged at13,200 rpm for 10 minutes and 4° C. and the supernatant was passedthrough a 0.45 μm filter. The filtrate was diluted 10-fold in methanol.Samples were analyzed by LC/MS/MS to monitor the D-tryptophan formed(see description below).

LC/MS/MS Method for Detecting D- and L-Tryptophan

LC/MS/MS screening was achieved by injecting samples from 96-well platesusing a CTCPal auto-sampler (LEAP Technologies, Carrboro, N.C.) into a70/30 MeOH/H₂O (0.25% AcOH) mixture provided by LC-10ADvp pumps(Shimadzu, Kyoto, Japan) at 0.8 mL/min through a Chirobiotic T column(4.6×250 mm) and into the API4000 Turbolon-Spray triple-quad massspectrometer (Applied Biosystems, Foster City, Calif.). Ion spray andMultiple Reaction Monitoring (MRM) were performed for the analytes ofinterest in the positive ion mode and each analysis lasted 15.0 minutes.D- and L-tryptophan parent/daughter ions: 205.16/188.20.

Example 5 Racemase Activity Dependent Upon Conditions

SEQ ID NO:402, SEQ ID NO:386, SEQ ID NO:396, SEQ ID NO:414, SEQ IDNO:420, SEQ ID NO:422, SEQ ID NO:122*, SEQ ID NO:438, SEQ ID NO:428**,SEQ ID NO:434, & SEQ ID NO:436 are racemase subclones that were activeunder the conditions described in Part A. These subclones were notactive under the conditions described in Part B (see Example 6 fordetails for details on SEQ ID NO:386, SEQ ID NO:396, SEQ ID NO:402; seeExample 7 for details on SEQ ID NO:414; see Example 12 for details onSEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:122, SEQ ID NO:428, SEQ IDNO:434, SEQ ID NO:436, SEQ ID NO:438).

The racemase subclones were in the pSE420-C-His vector/E. coli MB2946host (Strych & Benedik, 2002, J. Bacteriology, 184:4321-5) with theexception of SEQ ID NO:414. SEQ ID NO:414 was in pse420-c-His/E. coliTop10 host (Invitrogen, Carlsbad, Calif.). The his-tag was not expressedin any of these subclones.

The subclones were grown, lysed and lyophilized according to theprocedures described in Example 4. Samples were tested for activityusing a racemase assay (as described in Example 4). Racemases wereincubated with 10 mM L-tryptophan and 10 μM PLP at pH 8 and 37° C. Allracemases were loaded at 10 mg/mL total protein with the exception ofSEQ ID NO:402. SEQ ID NO:402 was loaded at 5 mg/mL total protein becausethere was not enough biomass to allow for a higher loading.

At indicated timepoints, 50 μL of the reaction product was added to 150μL of ice cold acetonitrile. Samples were vortexed for 30 seconds andthe supernatant was then diluted fifty-fold in methanol. Samples werethen analyzed by LC/MS/MS (as described in Example 4) to monitor theD-tryptophan formed and the residual L-tryptophan remaining.

Tables 10, 11, 12, and 13 show the racemase activity over time. Samplesthat were assayed together are grouped together in a single table.

TABLE 10 Racemase activity assay for SEQ ID NO: 414, SEQ ID NO: 420, SEQID NO: 422, SEQ ID NO: 434, and SEQ ID NO: 436, SEQ ID NO: 438 μg/mlD-tryptophan formed Time SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID No(−) (+) (hours) NO: 414 NO: 420 NO: 422 NO: 434 NO: 436 NO: 438 enzymecontrol control 0 0.19 0.96 0.96 0.20 0.18 22.35 0.18 0.26 0.23 2 33.23309.78 187.81 1.63 0.21 38.70 0.21 0.18 58.97 4 51.87 320.22 191.46 2.922.85 71.35 0.21 0.22 88.35 24 119.45 344.74 128.80 8.01 4.19 101.03 0.180.22 180.42

TABLE 11 Racemase activity assay for SEQ ID NO: 402 μg/ml D-tryptophanformed Time (hours) SEQ ID NO: 402 (−) control (+) control 0 0.00 1.858.29 2 19.67 0.00 583.79 4 18.25 0.00 715.26 24 44.05 4.32 730.14

TABLE 12 Racemase activity assay for SEQ ID NO: 396 μg/ml D-tryptophanformed Time (hours) SEQ ID NO: 396 (−) control (+) control 0 3.51 0.000.00 2 10.58 0.00 189.04 4 12.29 0.00 231.84 24 63.49 0.63 609.44

TABLE 13 Racemase activity assay for SEQ ID NO: 386 μg/ml D-tryptophanformed Time (hours) SEQ ID NO: 386 (−) control (+) control 0 0.00 0.000.80 2 0.00 0.00 189.04 4 6.03 0.00 230.98 24 5.23 3.10 478.75

In summary, SEQ ID NO:402, SEQ ID NO:386, SEQ ID NO:396, SEQ ID NO:414,SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:122*, SEQ ID NO:438, SEQ IDNO:434, and SEQ ID NO:436 were active on tryptophan under the conditionsdescribed in Part A. These samples were not active under the conditionsdescribed in Part B (see Examples 6, 7, and 12). The differences inobserved racemase activity may be attributed to differences in hoststrains, expression conditions, post-expression cell handling and assayprotein-loading. Refer to Example 3 for SEQ ID NO:122 activity data. Itis noted that SEQ ID NO:428 is not included here because it did notreach an inducible OD₆₀₀ and, therefore, was not induced.

It is expected that the presence of activity in a polypeptide encodedfrom a subcloned nucleic acid is predictive of the presence of activityin the corresponding polypeptide encoded from the full-length or wildtype nucleic acid.

TABLE 14 Sub-clone number Wild-Type Clone SEQ ID NO: 402 SEQ ID NO: 52SEQ ID NO: 386 SEQ ID NO: 36 SEQ ID NO: 396 SEQ ID NO: 10 SEQ ID NO: 414SEQ ID NO: 8 SEQ ID NO: 420 SEQ ID NO: 116 SEQ ID NO: 422 SEQ ID NO: 118SEQ ID NO: 122 SEQ ID NO: 122 SEQ ID NO: 438 SEQ ID NO: 104 SEQ ID NO:434 SEQ ID NO: 56 SEQ ID NO: 436 SEQ ID NO: 114

Part B Example 6 Analysis of Racemases Provided as pSE420 Clones

SEQ ID NO:386, SEQ ID NO:388, SEQ ID NO:390, SEQ ID NO:392, SEQ IDNO:394, SEQ ID NO:396, SEQ ID NO:398, SEQ ID NO:400, SEQ ID NO:402, SEQID NO:404, SEQ ID NO:406, SEQ ID NO:408, and SEQ ID NO:410 racemaseswere provided as pSE420 clones. One skilled in the art can synthesizethe genes encoding these racemases using various published techniquesfor example, as described in Stemmer et al., 1995, Gene, 164(1):49-53.The plasmids were transformed into E. coli XL-1 Blue (Novagen/EMDBiosciences, San Diego, Calif.) cells as per manufacturer instructions.

Transformants were grown overnight at 37° C. and 250 rpm in 5 ml LBcontaining ampicillin (100 μg/mL). Overnight cultures were used toinoculate 25 mL of the same media in 250 mL baffled shake flasks.Cultures were grown at 30° C. and 250 rpm until they reached an OD₆₀₀ of0.6, after which protein expression was induced with 1 mM IPTG for 4.25h at 30° C. Samples for total protein were taken prior to induction andright before harvesting. Cells were harvested by centrifugation andfrozen at −80° C.

TABLE 15 D-trp Production μg/mL D-trp μg/mL D-trp Racemase Candidate 2hours 21 hours pSE420 vector control nd nd SEQ ID NO: 386 nd nd SEQ IDNO: 388 nd nd SEQ ID NO: 390 nd nd SEQ ID NO: 392 nd nd SEQ ID NO: 394nd 6 SEQ ID NO: 396 nd nd SEQ ID NO: 398 nd nd SEQ ID NO: 414 nd nd SEQID NO: 400 46 410 SEQ ID NO: 402 nd nd SEQ ID NO: 404 10 18 SEQ ID NO:406 1171 2724 SEQ ID NO: 408 248 785 SEQ ID NO: 410 502 1435 A. caviaewild-type BAR 1695 3820 nd = not detected under the conditions of theassay as described above

It is noted that, when cell-free extracts were used, very low expressionwas observed. It was concluded, therefore, that the cell-free extractslikely contained significantly less protein than the purified positivecontrol enzyme (wild-type A. caviae)

Tryptophan racemase activity was detected for SEQ ID NO:400, SEQ IDNO:404, SEQ ID NO:406, SEQ ID NO:408 and SEQ ID NO:410 using theconditions described in Part B. Similar results were obtained for SEQ IDNO:400, SEQ ID NO:404, SEQ ID NO:406, SEQ ID NO:408 and SEQ ID NO:410using the reaction conditions described in Part A. In addition,detectable activity was observed for candidates SEQ ID NO:386, SEQ IDNO:396, and SEQ ID NO:402 using conditions described in Part A, but wasnot observed using conditions described in Part B (see, for example,Example 5). Detectable activity was not observed for SEQ ID NO:394 underthe conditions described in Part A, while very low activity (barelydetectable at 21 hours) was observed for SEQ ID NO:394 under theconditions described in Part B.

Some constructs were observed, under the conditions described in Part A,to be unstable in expression systems, particularly those with a leadersequence. The host organisms, expression conditions, and post expressioncell handling can all affect whether there is detectable tryptophanracemase activity under the conditions of the assay. Additionally, underoptimized conditions, it is expected that all racemase candidates couldhave tryptophan racemase activity.

The presence of activity in a polypeptide encoded from a subclonednucleic acid is predictive of (also demonstrates) the presence ofactivity in the corresponding polypeptide encoded from the full-lengthor wild type nucleic acid.

TABLE 16 Sub-clone number Wild-Type Clone SEQ ID NO: 400 SEQ ID NO: 42SEQ ID NO: 404 SEQ ID NO: 54 SEQ ID NO: 406 SEQ ID NO: 58 SEQ ID NO: 408SEQ ID NO: 48 SEQ ID NO: 410 SEQ ID NO: 46 SEQ ID NO: 394 SEQ ID NO: 4

Example 7 Characterization of Racemase SEQ ID NO:414 and Racemase SEQ IDNO:412

SEQ ID NO:412 and SEQ ID NO:414 were both found to be active whenassayed for tryptophan racemase activity under the conditions describedin Part A. One skilled in the art can synthesize the genes encodingthese racemases using various published techniques for example, asdescribed in Stemmer et al., supra. It should be noted that 10 mg oftotal protein in the form of lyophilized cell extracts was used in PartA when evaluating racemase activity (see Example 4). In some cases, thiswas ten times as much total soluble protein as was used in the assaysdescribed in Part B. This differences in the amount of protein used inthe assays (i.e., of Part A vs. Part B) may explain, at least in part,some of the differences in activity observed with the same polypeptide.

SEQ ID NO:414 was expressed in 3 different hosts in Part A (MB2946, XL-1Blue, and TOP10). High activity was observed in cell-free extract fromthe TOP10 host, with only a small amount of activity observed in XL-1Blue and no detectable product formed from the MB2946 host under theconditions of the assay. SEQ ID NO:412 was expressed in the MB2946 hostand found to be highly active.

SEQ ID NO:412 and SEQ ID NO:414 were received as pSE420 constructs,which were initially evaluated in E. coli TOP10. Strains were grown andinduced, and cell extracts were prepared as described in Part B.

Tryptophan racemase assays were carried out using desalted cell-freeextracts under the conditions described in Example 17.

Purified A. caviae D76N (100 μg) served as a positive control for theassay, and cell-free extract of E. coli host cells containing the emptyvector pSE420 served as a negative control. 1.4 mg of total protein wasused for SEQ ID NO:412 and SEQ ID NO:414.

TABLE 17 Trp Assay results D-trp production, μg/mL Time pSE420 (vectorA. caviae SEQ ID SEQ ID (Hr) control) D76N NO: 414 NO: 412 0 nd nd nd nd0.5  9 1797 nd 1205 1 12 3764 nd 1765 4 24 3818  2 3012 22 22 3621 332204 nd = not detected under the conditions of the assay as describedabove control was purified BAR from A. caviae D76N mutant - 100 μg/mlnot much activity detected in crude extract from SEQ ID NO: 414 (1.4mg/ml) some limited (very low) activity in extracts containing pSE420vector control considering that no band was observed in crude extractsfrom SEQ ID NO: 412, good activity.

There was very little activity detected in crude extract from SEQ IDNO:414 as well as negative control. SEQ ID NO:412 gave high specificactivity given that there was barely detectable protein band observed inthe soluble fraction (comparing 100 μg of purified A. caviae BAR to anestimated less than 30 μg of SEQ ID NO:412, assuming it was 2% or lessof the total protein).

The host organisms, expression conditions, and post expression cellhandling can all affect whether there is detectable tryptophan racemaseactivity under the conditions of the assay. Additionally, underoptimized conditions, it is expected that all racemase candidates couldhave tryptophan racemase activity.

It is expected that the presence of activity in a polypeptide encodedfrom a subcloned nucleic acid is predictive of the presence of activityin the corresponding polypeptide encoded from the full-length or wildtype nucleic acid.

TABLE 18 Sub-clone number Wild-Type Clone SEQ ID NO: 414 SEQ ID NO: 8SEQ ID NO: 412 SEQ ID NO: 62

Example 8 SEQ ID NO:412 is More Active on Tryptophan than Alanine

In order to get a more quantitative comparison of SEQ ID NO:412 to thebenchmark BAR (A. caviae D76N), SEQ ID NO:412 was PCR-amplified withNcoI and Xho I restriction sites for subcloning into pET28 (Novagen/EMDChemicals, San Diego, Calif.).

SEQ ID Designation Sequence NO: SEQ ID GGTTCCGGAACCATGGCCGAAACAAATCTGC503 NO: 412 F Nco1 SEQ ID GGTTCCAAGGCTCGAGCTATTTTTGTTCTGC 504 NO: 412 RTATTCTATATGTC Xho1 with stop SEQ ID GGTTCCAAGGCTCGAGTTTTTGTTCTGCTAT 505NO: 412 R TCTATATGTC Xho1

pET28 constructs were created with and without a C-terminal His tag(tagged constructs were created by using a reverse primer without a stopcodon in the PCR). pET26b constructs were created with a C-terminal Histag. Constructs were sequenced for accuracy (Agencourt Bioscience Inc.,Beverly Mass.) and used to transform E. coli BL21(DE3) (Novagen/EMDBiosciences, San Diego, Calif.).

Transformants were grown and induced in OvernightExpress™ media andcell-free extracts were prepared as described herein. Proteins werepurified from tagged constructs on Novagen/EMD Biosciences His-bindcolumns (Novagen/EMD Biosciences, San Diego, Calif.) and desalted onPD-10 columns; for untagged constructs, cell-free extracts were desaltedon PD-10 columns

Protein concentrations were determined by Pierce BCA protein assay andracemase purity was determined by Experion Automated Gel System(Experion, version A.01.10, Biorad, Hercules, Calif.). Racemase assayswere performed on purified and crude protein extracts as described inExample 17. Racemase expression in the pET26b construct was lower thanthe pET28 vector, however active SEQ ID NO:412 protein was obtained.Results for SEQ ID NO:412/pET28 are shown in this example.

D-trp production (μg/mL) in SEQ ID NO: 412 (NOT normalized) A. caviaeSEQ ID NO: 412/ SEQ ID NO: 412/ Time D76N pET28 CFE pET28 purified (Hr)(100 μg) (100 μg) (30 μg) 0 nd nd 70 0.5 1114 465 3133 1 3487 791 3350 24029 1113  4485 *Note 30 μg of SEQ ID NO: 412/pET28 purified protein wasused as compared to 100 μg of other enzyme preps. nd = not detectedunder the conditions of the assay as described above

Purified SEQ ID NO:412 protein from construct in pET28 was furthercharacterized for racemase activity on tryptophan, alanine, and monatin.Tryptophan, monatin, and alanine assays were performed as described inExample 17, with A. caviae D76N serving as positive control forracemization assays.

SEQ ID NO:412 Racemase Prefers Tryptophan as a Substrate

Time (min) A. caviae D76N SEQ ID NO: 412/pET28 purified D-Trpnmoles/μl/μg protein 0 nd nd 5 59 353 10 123 500 20 128 715 60 305 2145D-Ala nmoles/μl/μg protein 0 44 nd 5 1320 nd 10 2239 23 20 2602 314 604654 1044 nd = not detected under the conditions of the assay asdescribed above

SEQ ID NO:412 consistently gave higher D-trp activity than the controlracemase candidate, BAR, A. caviae D76N. SEQ ID NO:412 appears to bespecific for tryptophan versus alanine as a substrate for racemization.In contrast, A. caviae D76N BAR while active on tryptophan, has apreference for alanine as a substrate. The ability of purified SEQ IDNO:412 to racemize 7 additional L-amino acids was evaluated and thedetails are reported in Example 10.

In addition, the impact of alanine on tryptophan racemase activity wasinvestigated. An experiment was designed to determine the impact ofL-alanine on the racemization of L-tryptophan by either BAR A. caviaeD76N or racemase candidate SEQ ID NO:412. Racemase enzymes were assayedin the presence of tryptophan and alanine together to furthercharacterize substrate preference/competition. Assay was carried out asdescribed in Example 17, with 30 mM of each substrate (L-Trp and L-Ala)in the reaction. For both racemase enzymes, control racemase assays wereconducted in the presence of L-tryptophan alone. The data from thesecontrol assays at various time points were considered to be 100% whencompared with the respective data from assays with both amino acids.

Competition of L-Ala and L-Trp in Racemase Assay

% D-trp formed (100% without L-Ala assumed) A. caviae D76N SEQ ID SEQ IDNO: Time A. caviae (Trp only NO: 412pET28 412pET2 purified8 (min) D76Ncontrol) purified (Trp only control)  0 min 65% 100%  5 min   0% 100%39% 100% 20 min 54.6% 100% 96% 100% 60 min 36.7% 100% 94% 100% 180 min 36.4% 100% 110.6%   100%

Despite some initial inhibition of tryptophan racemization between zeroand five minutes, with SEQ ID NO:412 there was little to no impact ofL-alanine. SEQ ID NO:412 retained 96%-100% of its tryptophan racemaseactivity between 20 minutes to the end of the assay at three hours. Incontrast, BAR A. caviae D76N only retained 37%-55% of its tryptophanracemase activity in the presence of L-alanine, during the same timeperiod. Thus, the preference of SEQ ID NO:412 for tryptophan as asubstrate is advantageous in the presence of competing substrates likealanine.

Example 9 Racemases Lacking Monatin Racemization Activity

TABLE 19 SEQ ID NO: 412 and A. caviae D76N do not racemize monatinMonatin Isomer Ratio BAR Time % % % BAR ug Vol Ptn (hr) S,S R,S R,R A.caviae (D76N) 50 11.9 μL  0 1.0 0.9 98.1 SEQ ID NO: 412pET26b 50 73 μL 00.6 0.8 98.6 SEQ ID NO: 412pET28 50 98 μL 0 1.0 1.1 97.9 Negativecontrol 0 0 0 0.4 0.1 98.5 A. caviae (D76N) 50 24 0.6 1.0 98.4 SEQ IDNO: 412pET26b 50 24 0.5 1.0 98.5 SEQ ID NO: 412pET28 50 24 0.4 1.3 98.3Negative control 0 24 0.6 0.9 98.5 A. caviae (D76N) 50 48 0.4 0.7 98.9SEQ ID NO: 412pET26b 50 48 0.4 0.8 98.8 SEQ ID NO: 412pET28 50 48 1.30.5 98.2 Negative control 0 48 0.4 0.7 98.9

Neither SEQ ID NO:412 nor the benchmark A. caviae BAR showed detectableracemization of R,R monatin under the conditions of the assay asdescribed in Example 17.

TABLE 20 Racemase Substrate Specificity R,R Designation Alanine monatinVector/E. coli Host Reference, A. caviae +++ − pET30/BL21DE3 SEQ ID NO:412 − (low, prefers − pSE420/TOP10, trp) pET28/BL21DE3 *SEQ ID NO: 442 +− pSE420/TOP10 SEQ ID NO: 456 + − pSE420/TOP10 SEQ ID NO: 458 + −pSE420/TOP10 SEQ ID NO: 462 + − pSE420/TOP10 SEQ ID NO: 464 + −pSE420/TOP10 SEQ ID NO: 466 + − pSE420/TOP10 SEQ ID NO: 468 + −pSE420/TOP10 SEQ ID NO: 470 + − pSE420/TOP10 SEQ ID NO: 472 +++ −pSE420/TOP10 SEQ ID NO: 478 + − pSE420/TOP10 SEQ ID NO: 314 + −pET30/BL21DE3 SEQ ID NO: 326 + − pET30/BL21DE3 SEQ ID NO: 340 not tested− pET30/BL21DE3 SEQ ID NO: 342 + − pET30/BL21DE3 SEQ ID NO: 344 ? −pET30/BL21DE3 SEQ ID NO: 318 + − pET30/BL21DE3 SEQ ID NO: 330 + −pET30/BL21DE3 SEQ ID NO: 322 + − pET30/BL21DE3 SEQ ID NO: 324 + −pET30/BL21DE3 SEQ ID NO: 328 + − pET30/BL21DE3 SEQ ID NO: 346 + −pET30/BL21DE3 SEQ ID NO: 348 ? − pET30/BL21DE3 SEQ ID NO: 334 + −pET30/BL21DE3 SEQ ID NO: 350 not tested − pET30/BL21DE3 SEQ ID NO: 352 +− pET30/BL21DE3 − indicates no detectable racemization under theconditions of the assays after a minimum of 24 hours *indicates enzymesthat were re-cloned in pET30a with a C-terminal His tag for purificationand more quantitative assays

Example 10 SEQ ID NO:412 is a Broad Specificity Amino Acid Racemase

The ability of purified SEQ ID NO:412 to racemize 7 additional L-aminoacids was evaluated. The amino acid racemase assay was carried out asdescribed in Example 17, with 30 mM of each L-amino acid substrate andapproximately 1 μg of purified racemase candidate SEQ ID NO:412 (frompET28/BL21(DE3) induction) added for each amino acid substrate assayed.

TABLE 21 Additional substrates for SEQ ID NO: 412 μg/mL correspondingStarting substrate D-amino acid Relative Activity (L-amino acid)produced (2 hours) (Trp taken as 100%) Leucine 2429.2 131.8% Phenylalanine 2193.5  119% Tryptophan 1843.5  100% Methionine 1387.775.3% Tyrosine 154.1 8.36% Alanine 132.3 7.18% Lysine 49.3 2.67%Aspartic acid 23 1.25% Glutamate 1.9  0.1%

SEQ ID NO:412 appears to be an amino acid racemase with broad substratespecificity and seems to prefer bulky, hydrophobic amino acids.

Racemase activity for various amino acids as substrates was observed asfollows, under the conditions of the assay as described:[Leucine/Phenylalanine/Tryptophan/Methionine]>[Tyrosine/Alanine]>[Lysine/AsparticAcid]>Glutamate.

It should be noted that analytical methods for detection of all of theabove D-amino acids with the exception of tryptophan aresemi-quantitative so these results indicate a trend in racemaseactivity.

Example 11 Methods to Improve Solubility of an Insoluble Protein and itsActivity on Tryptophan

SEQ ID NO:412 showed lower solubility than other racemase candidatesdescribed in this application, under the expression conditions tested.The SEQ ID NO:412 insoluble protein fraction was tested for racemizationactivity on tryptophan.

Cell-free extracts of pET28/24431 were prepared from frozen cell pelletsby adding 5 ml of Bugbuster Amine Free (Novagen/EMD Biosciences, SanDiego, Calif.) with 5 μL/mL of Protease Inhibitor Cocktail II(Calbiochem, San Diego, Calif.) and 1 μl/ml of benzonase nuclease(Novagen/EMD Biosciences, San Diego, Calif.), per gm of cell pellet.Cell pellet suspensions were incubated at room temperature with gentlemixing for 15 min; cells pellets were spun out at 14000 rpm for 20 min(at 4° C.) and retained for assays.

Cell pellets containing insoluble SEQ ID NO:412 racemase were washedmultiple times in phosphate buffered saline to remove traces ofsupernatant containing soluble SEQ ID NO:412 protein fraction. Washedpellets were used in qualitative tryptophan assays (amount of protein inassay was not quantitated, rather a set volume of pellet resuspended inphosphate buffer was added to assay). The experiment was performedtwice, once with pellets that were washed four times, and the secondtime with frozen pellets that were thawed and washed an additional sixtimes. Tryptophan racemization assays were performed on the insolubleprotein suspension as described in Example 17.

TABLE 22 SEQ ID NO: 412 insoluble protein assays - active pellets Time(hr) pET28 - 2 uL pET28 - 20 uL A. D-trp production (μg/mL) in pelletswashed 10X and after freeze thaw 0 nd nd 0.16  44 353 1 137 2437 B.D-trp production (μg/mL) in pellets washed 4X with Phosphate buffer 0 nd19 0.16 153 681 1 831 2225 nd = not detected under the conditions of theassay as described above

SDS-PAGE analysis of cell pellets/insoluble protein fraction from theBugbuster protocol above, showed a predominant protein band at theexpected size (56.3 kD) for SEQ ID NO:412 racemase. Insoluble SEQ IDNO:412/pET28 protein fraction was observed to have tryptophan racemaseactivity. D-tryptophan production in the case of 20 μl samples wascomparable between the two trials. The variation observed in the case ofthe 2 μl samples could be attributed to the small volume and samplenature (insoluble protein suspension).

Preliminary investigations indicated that SEQ ID NO:412 is not amembrane associated protein, which might be a possibility given the lackof solubility but the presence of activity in SEQ ID NO:412.

Experiments to Improve Solubility of SEQ ID NO:412

Various host systems reported to improve soluble expression ofheterologous proteins were investigated in an effort to improve solubleexpression of SEQ ID NO:412 racemase:

E. coli KRX (Promega, Madison, Wis.), CopyCutter™ EPI400™ (EpicentreBiotechnologies, Madison, Wis.), ArcticExpress™ (Stratagene, La Jolla,Calif.), E. coli HMS174 (Novagen/EMD Biosciences, San Diego, Calif.),and E. coli EE2D.

A. Induction in ArcticExpress™

Competent cells of ArcticExpress™ (DE3) were transformed withpET28/24431 and pET26b/24431 as per manufacturer's protocol (Stratagene,La Jolla, Calif.).

Transformants were grown in LB containing kanamycin (50 mg/L) andgentamycin (20 mg/L) overnight at 37° C. and 250 rpm. A 2% inoculum wastransferred to 50 mL OVERNIGHTEXPRESS™ media containing kanamycin andgentamycin. Flasks were grown for 1.5 days at 15° C. and 250 rpm. Cellswere harvested and cell extracts prepared as described in herein.SDS-PAGE analysis of total and soluble protein was conducted.

No improvement was seen in solubility in the ARCTICEXPRESS™ strain.However, the chaperonin proteins that should be overexpressed in thisstrain were not observed (expected sizes of 10 kDa and 60 kDa) on theSDS-PAGE gel. The experiment was repeated with fresh competent cells andinduction over 3 days, but SDS results were identical.

When the ARCTICEXPRESS™ experiments were repeated with the pET28/24431construct using the methods of Part A, the data showed an improvement insoluble protein expression (see Example 1).

B. Induction in E. coli Copycutter™

COPYCUTTER EPI400™ cells were transformed with pET28/SEQ ID NO:412 asper manufacturer instructions (Epicentre Biotechnologies, Madison,Wis.). Liquid cultures of transformants were grown overnight (LBkanamycin 50, 37° C., 250 rpm) and used to inoculate shake flaskscontaining 25 mL LB media, kanamycin (50 mg/L) and 1× CopyCutter™induction solution. Cultures were grown at 30° C. and 250 rpm for 5hours. Cultures were harvested and cell extracts were prepared asdescribed herein. SDS-PAGE analysis of total and soluble protein wasconducted.

C. Induction in E. coli HMS 174 and EE2D DE3

E. coli HMS 174 (Novagen/EMD Biosciences, San Diego, Calif.) and E. coliBW30384(DE3)-ompT-metE (“E. coli EE2D”) competent cells were transformedwith pET28/SEQ ID NO:412. (Construction of the E. coliBW30384(DE3)-ompT-metE expression host and the transformation protocolare described in WO 2006/066072. Liquid cultures of transformants weregrown overnight (LB kanamycin 50, 37° C., 250 rpm) and used to inoculate50 mL shake flasks of overnight express media containing kanamycin (50mg/L). Cultures were grown at 30° C. and 250 to an OD₆₀₀>10. Cultureswere harvested and cell extracts were prepared as described herein.SDS-PAGE analysis of total and soluble protein was conducted.

In all cases described above, no significant increase in solubleexpression of SEQ ID NO:412 was observed based on SDS-PAGE analyses. Inaddition, SEQ ID NO:412 was subcloned into a derivative of the pET23dvector (Novagen, Madison, Wis.) containing the E. coli metE gene andpromoter inserted at the NgoMIV restriction site and a second psilrestriction site that was added for facile removal of the beta-lactamasegene (bla). The construction of this vector is described in WO2006/066072. This construct was transformed into E. coli B834 DE3 hostsystem (Novagen/EMD Biosciences, San Diego, Calif.), without significantincrease in soluble expression.

Since SEQ ID NO:412 with its native leader sequence could not besuccessfully cloned and propagated under the conditions described inPart A, a N-terminal alanine residue was added in place of the nativeleader sequence of SEQ ID NO:412. It was determined that deletion ofthis additional alanine residue had no impact on soluble expression,based on SDS-PAGE analysis.

The presence of DTT was shown to minimize protein precipitation duringpurification of selected histidine-tagged D-aminotransferase candidates.The addition of 5 mM DTT during the bugbuster solubilization andsubsequent purification of histidine-tagged SEQ ID NO:412 frompET28/BL21DE3 induction did not impact soluble expression as observed onSDS-PAGE.

One skilled in the art could employ various methods reported in theliterature to improve soluble expression of the protein.

Example 12 Analysis of Racemases Provided as pSE420 Clones

SEQ ID NO:412, SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:420, SEQ IDNO:422, SEQ ID NO:424, SEQ ID NO:122, SEQ ID NO:428, SEQ ID NO:430, SEQID NO:432, SEQ ID NO:434, SEQ ID NO:436, SEQ ID NO:438 and SEQ ID NO:440racemases were provided as pSE420 clones. One skilled in the art couldsynthesize the genes encoding these racemases using various publishedtechniques for example, as described in Stemmer et al., supra. Theplasmids were transformed into E. coli TOP10 chemically competent cells(Invitrogen, Carlsbad, Calif.). Overnight cultures grown in LBcarbenicillin (100 μg/ml) were diluted a hundred-fold in 50 ml LBcarbenicillin (100 μg/ml) in a 250 ml baffled flask. Cultures were grownat 30° C. with agitation at 250 rpm until they reached an OD₆₀₀ of 0.5to 0.8, after which protein expression was induced with 1 mM IPTG for 4h at 30° C. Samples for total protein were taken prior to induction andright before harvesting. Cells were harvested by centrifugation. Cellswere frozen at −80° C.

Cell extracts were typically prepared from the above frozen pellets byadding 5 ml per g of cell pellet of Bugbuster Amine Free (Novagen/EMDBiosciences, San Diego, Calif.) with 5 μL/mL of Protease InhibitorCocktail II (Calbiochem, San Diego, Calif.) and 1 μl/ml of benzonasenuclease (Novagen/EMD Biosciences, San Diego, Calif.). Cell solutionswere incubated at room temperature with gentle mixing for 15 min; cellswere spun out at 14000 rpm for 20 mM (at 4° C.) and the supernatant wascarefully removed. Detergents and low molecular weight molecules wereremoved by passage through PD-10 columns (GE Healthcare, Piscataway,N.J.) previously equilibrated with 100 mM potassium phosphate (pH 7.8)with 0.05 mM PLP. Proteins were eluted with 3.5 mL of the same buffer.Total protein concentration was determined using the Pierce BCA totalprotein assay with bovine serum albumin (BSA) as the standard, per themanufacturer's instructions (Pierce Biotechnology, Inc., Rockford,Ill.). The resulting cell-free extract was used for subsequent assays.

For the tryptophan racemase assay a total of 650 μg of desalted proteinwas added for each enzyme based on Pierce BCA total protein analysiswith BSA as the standard (Pierce Biotechnology, Inc., Rockford, Ill.).Formation of D-tryptophan was measured at 30 minutes, 2 hours, 4 hoursand 24 hours. pSE420 cell-free extract of SEQ ID NO:412 served as apositive control for the assay, and cell-free extract of empty vectorpSE420 served as a negative control.

TABLE 23 D-trp production, (pSE420 constructs) D-trp production, μg/mLEnzyme 30 min 2 hours 4 hours 24 hours pSE420 vector control nd nd nd ndSEQ ID NO: 412 25  79 126  582 SEQ ID NO: 416 82 336 568 2344 SEQ ID NO:418 59 209 307 1346 SEQ ID NO: 420 nd nd nd nd SEQ ID NO: 422 nd nd ndnd SEQ ID NO: 424 1407  *no data 3322  2564 SEQ ID NO: 122 nd nd nd ndSEQ ID NO: 428 nd nd nd nd SEQ ID NO: 430 nd nd nd nd SEQ ID NO: 432 ndnd nd nd SEQ ID NO: 434 nd nd nd nd SEQ ID NO: 436 nd nd nd nd SEQ IDNO: 438 nd nd nd nd SEQ ID NO: 440 864  2213  3022  3947 nd = notdetected under the conditions of the assay as described above *samplewas not tested

Racemases SEQ ID NO:420, SEQ ID NO:422, SEQ ID NO:122, SEQ ID NO:428,SEQ ID NO:430, SEQ ID NO:432, SEQ ID NO:434, SEQ ID NO:436, and SEQ IDNO:438 showed no detectable tryptophan racemase activity after 24 hoursunder the conditions tested. (Under the conditions described in Part A,good activity was observed for SEQ ID NO:420, SEQ ID NO:422, SEQ IDNO:122, and SEQ ID NO:438; very slight activity was detected for SEQ IDNO:428, SEQ ID NO:434, and SEQ ID NO:436; and no activity was detectedfor SEQ ID NO:440).

Racemases SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:424 and SEQ ID NO:440showed appreciable tryptophan activity in this assay. These were PCRamplified with and without C-terminal His tags for subcloning intopET30a. The oligonucleotides used for amplification are shown in Table24.

TABLE 24  Oligonucleotide primers SEQ ID Primer NO: SEQ IDGGCCTTAACTCGAGGCGGTTGATCTTCTTGGGGTTG  506: NO: 416 R Xho1 no stop SEQ IDGGCCTTAACATATGGCTCCCTATCTGCCCCTTGTGAC 507 NO: 416 F Nde1 SEQ IDCCTTGGAACTCGAGTTAGCGGTTGATCTTCTTGG 508 NO: 416 R Xhol no stop SEQ IDGGCCTTAACATATGGCCCCTTACCTGCCGCTG 509 NO: 418 F Nde1 SEQ IDCCGGAACCTTGGAACCGTCGACTTAGCGTTTGATCTTCTTGGG 510 NO: 418 R Sal 1 withstop SEQ ID GGCCTTAACCTTGTCGACGCGTTTGATCTTCTTGGGGTTGGTGTAG 511 NO: 418 RSal 1 no stop SEQ ID GGCCTTAACATATGGCTCCACCGCTGTCGATGGACAAC 512NO: 424 F Nde1 SEQ ID GGCCTTAACATATGGCTAGCAATGCCTGGGTGGAGATAGAC 513NO: 440 F Nde1 SEQ ID GGCCTTAACTCGAGTTAGGTGTTGCCCCAGACGGTGTAC 514NO: 440 R Xho1 with stop SEQ IDGGCCTTAACTCGAGGGTGTTGCCCCAGACGGTGTACATGTCC 515 NO: 440 R Xho1 no stop

Tagged and untagged constructs were sequenced for accuracy (AgencourtBioscience Inc., Beverly Mass.) and transformed into BL21DE3;transformants were grown and induced in Overnight Express media andcell-free extracts were prepared as described herein. Racemase candidateproteins were purified from tagged constructs and desalted on PD-10columns Untagged racemase candidate cell-free extracts were desalted onPD-10 columns Protein concentrations were determined by Pierce BCAprotein assay (Pierce Biotechnology, Inc., Rockford, Ill.) and racemasepurity was estimated by Experion Automated Gel System (Experion, versionA.01.10, Biorad, Hercules, Calif.).

Racemase assays were performed on purified and crude protein extracts asdescribed in Example 17. Purified SEQ ID NO:412 protein served as apositive control. For the assay 5 μg of equivalent BAR protein was addedfor the positive control SEQ ID NO:412, and an estimated 50 μgequivalent BAR protein was added for each of the other enzymes based onPierce BCA total protein analysis and racemase purity estimation byExperion Automated Gel System (Experion, version A.01.10™, Biorad,Hercules, Calif.).

TABLE 25 D-trp production D-trp production, μg/mL Enzyme 0.5 hr 2 hr 4hr SEQ ID NO: 412pET28 purified 2197  2431  3186  SEQ ID NO: 416 pET30purified 52 54 134 SEQ ID NO: 418pET30 purified 187  533  664 SEQ ID NO:424 pET30 purified nd nd nd SEQ ID NO: 440 pET30 purified nd nd nd SEQID NO: 416 CFE nd 84 100 SEQ ID NO: 418 CFE 36 82  74 SEQ ID NO: 424 CFEnd nd nd SEQ ID NO: 440 CFE nd nd nd nd = not detected under theconditions of the assay as described above 4 candidates that appeared tohave higher activity than SEQ ID NO: 412-SEQ ID NO: 416, SEQ ID NO: 418,SEQ ID NO: 424, SEQ ID NO: 440 not replicated in pET30 with BL21DE3 hostall experiments conducted with purified protein (approx 50 μg BAR)Previously shown in Part A that there are striking differences inactivity when the same construct in three different host backgrounds.

Extracts of SEQ ID NO:416, SEQ ID NO:418, SEQ ID NO:424 and SEQ IDNO:440 in pSE420/TOP10 exhibited tryptophan racemase activity, whileextracts from the same clones in pET30/BL21DE3 did not exhibit orexhibited very little tryptophan racemase activity. SEQ ID NO:424 andSEQ ID NO:440 showed no detectable tryptophan racemase activity inpurified or crude cell extracts when cloned into pET30 and expressed inE. coli BL21DE3, under the conditions tested. SEQ ID NO:416 and SEQ IDNO:418 showed tryptophan racemase activity for both purified and crudeextracts.

Since variations in racemase activity were observed with SEQ ID NO:416,SEQ ID NO:418, SEQ ID NO:424 and SEQ ID NO:440 in different vector andhost backgrounds, the reproducibility in the original pSE420 vector wasinvestigated. [It is noted that the SEQ ID NO:424 racemase candidatecould not be revived from glycerol stocks.] Racemase assay was repeatedusing 1 mg total protein (from pSE420/TOP10 cell-free extracts) of SEQID NO:416, SEQ ID NO:418 and SEQ ID NO:440 (and same assay conditions asthe original assay—results shown in FIG. 10. The 3 clones showedseverely diminished racemase activity (see FIG. 12). Comparison of theracemase activity for SEQ ID NO:416, SEQ ID NO:418 and SEQ ID NO:440 inFIGS. 10 and 12 shows that inconsistent results were obtained despiteusing the same vector/host background.

Conditions described under Part A resulted in similar observations ofclone/construct instability of a few of the racemase candidates.

TABLE 26 D-trp production D-trp production, μg/mL Enzyme 0.5 hr 2 hr 4hr pSE420 vector control nd nd nd SEQ ID NO: 412 636  1672 3154 SEQ IDNO: 416 11 nd  78 SEQ ID NO: 418 31  139  187 SEQ ID NO: 440 nd   2 ndnd = not detected under the conditions of the assay as described above

The host organisms, expression conditions, and post expression cellhandling can all affect whether there is detectable tryptophan racemaseactivity under the conditions of the assay. Additionally, underoptimized conditions, it is expected that all racemase candidates couldhave tryptophan racemase activity.

Racemase candidates were grouped by amino acid sequence homology, withclusters having 95% or greater homology at amino acid level to areference sequence. One or more representatives was/were chosen fromeach group for characterization of tryptophan racemase activity underthe conditions described in Part B.

Using SEQ ID NO:110 as the reference sequence, the following racemasecandidates had 97% or greater identity at amino acid level to the abovereference sequence: SEQ ID NO:136, SEQ ID NO:174, SEQ ID NO:138, SEQ IDNO:296. SEQ ID NO:416 is a non-leadered version of the reference SEQ IDNO:110 sequence. Under the conditions described in Part B (see, forexample, Example 17), tryptophan racemase activity was detected for thenon-leadered version (SEQ ID NO:416) of the reference candidate, SEQ IDNO:110. Thus, it would be expected that other racemase candidates with97% or greater sequence identity at the amino acid level would also havetryptophan racemase activity.

Using SEQ ID NO:116 as the reference sequence, the following racemasecandidates had 97% or greater identity at amino acid level to the abovereference sequence: SEQ ID NO:150, SEQ ID NO:192, SEQ ID NO:152, SEQ IDNO:118, SEQ ID NO:194, SEQ ID NO:154, SEQ ID NO:196, SEQ ID NO:158, SEQID NO:160. SEQ ID NO:420 is a non-leadered version of the reference SEQID NO:116 sequence. SEQ ID NO:422 is a non-leadered version of thereference SEQ ID NO:118 sequence. Under the conditions described in PartB (e.g., Example 17), tryptophan racemase activity was not detected forSEQ ID NO:420 and SEQ ID NO:422 which are the non-leadered versions ofSEQ ID NO:116 and SEQ ID NO:118 respectively—however, activity was notobserved under the assay conditions described in Part A. The hostorganisms, expression conditions, and post-expression cell handling canall affect whether there is detectable tryptophan racemase activityunder the conditions of the assay. Additionally, under optimizedconditions or as shown in the assay conditions described in Part A, itis expected that all the above racemase candidates could have tryptophanracemase activity.

It is expected that the presence of activity in a polypeptide encodedfrom a subcloned nucleic acid is predictive of the presence of activityin the corresponding polypeptide encoded from the full-length or wildtype nucleic acid.

TABLE 27 Sub-clone number Wild-Type Clone SEQ ID NO: 416 SEQ ID NO: 110SEQ ID NO: 418 SEQ ID NO: 112 SEQ ID NO: 424 SEQ ID NO: 50 SEQ ID NO:440 SEQ ID NO: 440

Example 13 Analysis of Racemases Provided as pSE420 Clones

SEQ ID NO:442, SEQ ID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ IDNO:450, SEQ ID NO:452, and SEQ ID NO:454 racemases were provided aspSE420 clones. One skilled in the art can synthesize the genes encodingthese racemases using various published techniques for example, asdescribed in Stemmer et al., supra. The plasmids were transformed intoTOP10-chemically competent cells (Invitrogen, Carlsbad, Calif.).Overnight cultures growing in LB carbenicillin (100 μg/ml) were diluted100× in 50 ml LB carbenicillin in a 250 ml baffled flask. Cultures weregrown at 30° C. and 250 rpm until they reached an OD₆₀₀ of 0.5 to 0.8,after which protein expression was induced with 1 mM IPTG for 4 h at 30°C. Samples for total protein were taken prior to induction and rightbefore harvesting. Cells were harvested by centrifugation. Cells werefrozen at −80° C.

Cell extracts were typically prepared from the above frozen pellets byadding 5 ml per g of cell pellet of Bugbuster Amine Free (Novagen/EMDBiosciences, San Diego, Calif.) with 5 μL/mL of Protease InhibitorCocktail II (Calbiochem, San Diego, Calif.) and 1 μl/ml of benzonasenuclease (Novagen/EMD Biosciences, San Diego, Calif.). Cell solutionswere incubated at room temperature with gentle mixing for 15 min; cellswere spun out at 14000 rpm for 20 min (at 4° C.) and the supernatant wascarefully removed. Detergents and low molecular weight molecules wereremoved by passage through PD-10 columns (GE Healthcare, Piscataway,N.J.) previously equilibrated with 100 mM potassium phosphate (pH 7.8)with 0.05 mM PLP. Proteins were eluted with 3.5 mL of the same buffer.Total protein concentration was determined using the Pierce BCA proteinassay (Pierce Biotechnology, Inc., Rockford, Ill.) with bovine serumalbumin (BSA) as the standard, per the manufacturer's instructions. Theresulting cell-free extract was used for subsequent assays.

Tryptophan racemase assays were carried out under the conditionsdescribed in Example 17. For the tryptophan racemization assay, a totalof 1 mg of soluble protein (based on Pierce BCA total protein analysiswith BSA as the standard) was added for each racemase candidate andpositive controls. pSE420/TOP10 cell-free extract of SEQ ID NO:412served as positive control for the assay, and cell-free extract of E.coli TOP10 (Invitrogen, Carlsbad, Calif.) containing vector pSE420served as a negative control. Total protein concentration was determinedusing the Pierce BCA protein assay (Pierce Biotechnology, Inc.,Rockford, Ill.) with bovine serum albumin (BSA) as the standard, per themanufacturer's instructions. Formation of D-tryptophan was measured at30 minutes, 2 hours and 4 hours as described in Example 18.

TABLE 28 D-trp production D-trp production, μg/mL Enzyme 0.5 hr 2 hr 4hr pSE420 vector control nd nd nd SEQ ID NO: 412 636 1672 3154 SEQ IDNO: 442 678 2107 3346 SEQ ID NO: 444 105 457 758 SEQ ID NO: 446 312 15491934 SEQ ID NO: 448 6 42 nd SEQ ID NO: 450 23 66 33 SEQ ID NO: 452 32 3685 SEQ ID NO: 454 257 577 888 nd = not detected under the conditions ofthe assay as described above

All of the racemase candidate extracts tested above, SEQ ID NO:442, SEQID NO:444, SEQ ID NO:446, SEQ ID NO:448, SEQ ID NO:450, SEQ ID NO:452and SEQ ID NO:454, had detectable tryptophan racemase activity under theconditions described above. In addition, tryptophan racemase activitywas detected for the positive control SEQ ID NO:412 extract and therewas no detectable activity in the case of the pSE420 vector controlextracts. It is expected that the homologs of the representativeracemase candidates having 95% or greater homology at amino acid level(see Table 29) will also have tryptophan racemase activity.

TABLE 29 Enzyme 0.5 hr 2 hr 4 hr pSE420 vector control nd nd nd SEQ IDNO: 412 636 1672 3154 SEQ ID NO: 442 678 2107 3346 SEQ ID NO: 444 105457 758 SEQ ID NO: 446 312 1549 1934 SEQ ID NO: 448 6 42 nd SEQ ID NO:450 23 66 33 SEQ ID NO: 452 32 36 85 SEQ ID NO: 454 257 577 888 nd, notdetected under the conditions of the assay as described above

Racemase candidates described in this example were grouped by amino acidsequence homology with clusters having 95% or greater homology at aminoacid level to a reference sequence. One or more representatives werechosen from each group for characterization of tryptophan racemaseactivity using the conditions described in Part B. Using SEQ ID NO:244as the reference sequence, the following racemase candidates had 97% orgreater identity at amino acid level to the above reference sequence:SEQ ID NO:248, SEQ ID NO:236, SEQ ID NO:246, SEQ ID NO:252, SEQ IDNO:250, and SEQ ID NO:254. SEQ ID NO:448 is a non-leadered version ofthe reference SEQ ID NO:244 sequence. Under the conditions described inPart B (e.g., Example 17), tryptophan racemase activity was detected forthe non-leadered version (SEQ ID NO:448) of the reference candidate, SEQID NO:244; as well as the non-leadered version (SEQ ID NO:450) of thecandidate, SEQ ID NO:248. Thus, it would be expected that other racemasecandidates with 97% or greater sequence identity at the amino acid levelwould also have tryptophan racemase activity.

Using SEQ ID NO:288 as a reference sequence, the following racemasecandidates had 97% or greater identity at amino acid level to the abovereference sequence: SEQ ID NO:274, SEQ ID NO:234, SEQ ID NO:220, SEQ IDNO:222, SEQ ID NO:226, SEQ ID NO:232, SEQ ID NO:240, SEQ ID NO:242, SEQID NO:258, SEQ ID NO:260, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:286,SEQ ID NO:290, SEQ ID NO:170, and SEQ ID NO:216. SEQ ID NO:454 is anon-leadered version of the reference SEQ ID NO:288 sequence; SEQ IDNO:452 is a non-leadered version of SEQ ID NO:274 sequence; and SEQ IDNO:446 is a non-leadered version of SEQ ID NO:234 sequence. Under theconditions of the assay as described in Example 17, tryptophan racemaseactivity was detected for the non-leadered version (SEQ ID NO:454) ofthe reference candidate, SEQ ID NO:288; as well as the non-leaderedversions (SEQ ID NO:452 and SEQ ID NO:446) of racemase candidates SEQ IDNO:274 and SEQ ID NO:234, respectively. Thus, it would be expected thatother racemase candidates listed above with 97% or greater sequenceidentity at the amino acid level would also have tryptophan racemaseactivity.

Using SEQ ID NO:218 as a reference sequence, the following racemasecandidates had 97% or greater identity at amino acid level to the abovereference sequence: SEQ ID NO:208, SEQ ID NO:210, SEQ ID NO:228, SEQ IDNO:230, SEQ ID NO:270, SEQ ID NO:272, SEQ ID NO:278, SEQ ID NO:280, SEQID NO:282, SEQ ID NO:284, SEQ ID NO:292, SEQ ID NO:198, SEQ ID NO:212,SEQ ID NO:214, and SEQ ID NO:114. SEQ ID NO:204 had 96% identity withSEQ ID NO:218 reference sequence. SEQ ID NO:444 is a non-leaderedversion of the reference SEQ ID NO:218 sequence. Under the conditionsdescribed in Part B (e.g., Example 17), tryptophan racemase activity wasdetected for the non-leadered version (SEQ ID NO:444) of the referencecandidate, SEQ ID NO:218. Thus it would be expected that other racemasecandidates with 97% or greater sequence identity at the amino acid levelwould also have tryptophan racemase activity.

SEQ ID NO:436 is a non-leadered version of SEQ ID NO:114 sequence. Underthe conditions of the assays described in Part B, tryptophan racemaseactivity was not detected for the non-leadered version (SEQ ID NO:436)of the racemase candidate SEQ ID NO:114, as shown in Example 12.

SEQ ID NO:442 was Subcloned into pET30a with a C-Terminal His Tag

A D56N mutant (corresponding to D76N mutation in A. caviae) was createdin SEQ ID NO:442. Mutagenesis was done using the QuickChange-Multisite-directed mutagenesis kit (Stratagene, La Jolla, Calif.), using theC-tagged SEQ ID NO:442 gene in pET30a as template. The followingmutagenic primer was used to make the D56N change as described inExample 19: 5′-CGCCATCATGAAGGCGAACGCCTACGGTCACG-3′ (SEQ ID NO:516).

The site-directed mutagenesis was done as described in themanufacturer's protocol. The resulting mutation was detrimental totryptophan racemase activity in this candidate.

It is expected that the presence of activity in a polypeptide encodedfrom a subcloned nucleic acid is predictive of the presence of activityin the corresponding polypeptide encoded from the full-length or wildtype nucleic acid.

TABLE 30 Sub-clone number Wild-Type Clone SEQ ID NO: 442 SEQ ID NO: 224SEQ ID NO: 444 SEQ ID NO: 218 SEQ ID NO: 446 SEQ ID NO: 234 SEQ ID NO:448 SEQ ID NO: 244 SEQ ID NO: 450 SEQ ID NO: 248 SEQ ID NO: 452 SEQ IDNO: 274 SEQ ID NO: 454 SEQ ID NO: 288

Example 14 Analysis of Racemases Provided as pSE420 Clones

SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ IDNO:464, SEQ ID NO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQID NO:474, SEQ ID NO:476, and SEQ ID NO:478 racemases were provided aspSE420 clones. One skilled in the art can synthesize the genes encodingthese racemases using various published techniques for example, asdescribed in Stemmer et al., supra. The plasmids were transformed intoE. coli TOP10 chemically competent cells (Invitrogen, Carlsbad, Calif.).Overnight cultures growing in LB carbenicillin (100 μg/ml) were diluted100× in 50 ml LB carbenicillin (100 μg/ml) in a 250 ml baffled flask.Cultures were grown at 30° C. at 250 rpm until they reached an OD₆₀₀ of0.5 to 0.8, after which protein expression was induced with 1 mM IPTGfor 4 h at 30° C. Samples for total protein were taken prior toinduction and right before harvesting. Cells were harvested bycentrifugation and frozen at −80° C.

Cell extracts were typically prepared from the above frozen pellets byadding 5 ml per g of cell pellet of Bugbuster Amine Free (Novagen/EMDBiosciences, San Diego, Calif.) with 5 μL/mL of Protease InhibitorCocktail II (Calbiochem, San Diego, Calif.) and 1 μl/ml of benzonasenuclease (Novagen/EMD Biosciences, San Diego, Calif.). Cell solutionswere incubated at room temperature with gentle mixing for 15 min; cellswere spun out at 14,000 rpm for 20 min (at 4° C.) and the supernatantwas carefully removed. Detergents and low molecular weight moleculeswere removed by passage through PD-10 columns (GE Healthcare,Piscataway, N.J.) previously equilibrated with 100 mM potassiumphosphate (pH 7.8) with 0.05 mM PLP. Proteins were eluted with 3.5 mL ofthe same buffer. Total protein concentration was determined using thePierce BCA (Pierce Biotechnology, Inc., Rockford, Ill.) protein assaywith bovine serum albumin (BSA) as the standard, per the manufacturer'sinstructions. The resulting cell-free extract was used for subsequentassays.

Desalted cell-free extracts of racemase candidates SEQ ID NO:456, SEQ IDNO:458, SEQ ID NO:460, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQID NO:468, SEQ ID NO:470, SEQ ID NO:472, SEQ ID NO:474, SEQ ID NO:476,SEQ ID NO:478 were prepared as described in other examples.

Tryptophan racemase assays were carried out under the conditionsdescribed in Example 17. For the tryptophan racemization assay a totalof 800 μg of soluble protein was added for each racemase candidate andpositive controls. pSE420/TOP10 cell-free extracts of SEQ ID NO:412 andSEQ ID NO:442 served as positive controls for the assay, and cell-freeextract of E. coli TOP10 (Invitrogen, Carlsbad, Calif.) containingvector pSE420 served as a negative control. Total protein concentrationwas determined using the Pierce BCA (Pierce Biotechnology, Inc.,Rockford, Ill.) protein assay with bovine serum albumin (BSA) as thestandard, per the manufacturer's instructions. Formation of D-tryptophanwas measured at 30 minutes, 2 hours and 4 hours as described in Example18.

TABLE 31 D-trp production D-trp production, μg/mL Enzyme 0.5 hr 2 hr 4hr pSE420 nd nd nd SEQ ID NO: 412 834 1861 2803 SEQ ID NO: 442 911 18632912 SEQ ID NO: 456 nd nd nd SEQ ID NO: 458 nd nd nd SEQ ID NO: 460  11  5  105 SEQ ID NO: 462 nd nd nd SEQ ID NO: 464 nd nd nd SEQ ID NO: 466nd nd nd SEQ ID NO: 468 nd nd nd SEQ ID NO: 470 nd nd nd SEQ ID NO: 472nd nd nd SEQ ID NO: 474 121  455 1101 SEQ ID NO: 476  28  100  249 SEQID NO: 478 nd nd nd nd = not detected under the conditions of the assayas described.

Racemase candidates SEQ ID NO:460, SEQ ID NO:474 and SEQ ID NO:476showed tryptophan racemase activity. Racemases SEQ ID NO:456, SEQ IDNO:458, SEQ ID NO:462, SEQ ID NO:464, SEQ ID NO:466, SEQ ID NO:468, SEQID NO:470, SEQ ID NO:472 and SEQ ID NO:478 showed no detectabletryptophan racemase activity after 4 hours under the conditions tested.In a follow up experiment, a 24-hour sample was evaluated forD-tryptophan production. None of the racemases listed above showeddetectable tryptophan racemase activity at 24 hours under the conditionsdescribed above. Of the candidates for which no activity was observed,SEQ ID NO:456, SEQ ID NO:458, SEQ ID NO:462, SEQ ID NO:464, SEQ IDNO:466, SEQ ID NO:468, SEQ ID NO:470, SEQ ID NO:472 and SEQ ID NO:478exhibited poor or questionable soluble protein expression. The hostorganisms, expression conditions, and post expression cell handling canall affect whether there is detectable tryptophan racemase activityunder the conditions of the assay. Additionally, under optimizedconditions, it is expected that all racemase candidates could havetryptophan racemase activity.

Racemase candidates were grouped by amino acid sequence homology, withclusters having 95% or greater homology at amino acid level to areference sequence. One or more representatives was/were chosen fromeach group for characterization of tryptophan racemase activity usingthe conditions described in Part B.

Using SEQ ID NO:108 as the reference sequence, the following racemasecandidates had 96% or greater identity at amino acid level to the abovereference sequence: SEQ ID NO:172, SEQ ID NO:178, SEQ ID NO:180, SEQ IDNO:182, SEQ ID NO:184, SEQ ID NO:140, SEQ ID NO:144, SEQ ID NO:188, SEQID NO:190, SEQ ID NO:112, SEQ ID NO:148, SEQ ID NO:156, SEQ ID NO:120and SEQ ID NO:162. SEQ ID NO:474 is a non-leadered version of thereference SEQ ID NO:108 sequence. Under the conditions described in PartB (e.g., Example 17), tryptophan racemase activity was detected for thenon-leadered version (SEQ ID NO:474) of the reference candidate, SEQ IDNO:108, as well as the non-leadered version (SEQ ID NO:460) of SEQ IDNO:120 which is 97% identical with the reference candidate, SEQ IDNO:108. Additionally the non-leadered version (SEQ ID NO:418) of SEQ IDNO:112 was shown to have detectable tryptophan racemase activity as seenin Example 12. Thus it would be expected that the other racemasecandidates listed above, with 96% or greater sequence identity at theamino acid level would also have tryptophan racemase activity.

It is expected that the presence of activity in a polypeptide encodedfrom a subcloned nucleic acid is predictive of the presence of activityin the corresponding polypeptide encoded from the full-length or wildtype nucleic acid.

TABLE 32 Sub-clone number Wild-Type Clone SEQ ID NO: 460 SEQ ID NO: 120SEQ ID NO: 474 SEQ ID NO: 108 SEQ ID NO: 476 SEQ ID NO: 300

Example 15 Analysis of Racemase Candidates Provided as PCR Products

First Group

Racemases SEQ ID NO:314, SEQ ID NO:326, SEQ ID NO:342, SEQ ID NO:344,SEQ ID NO:318, SEQ ID NO:330, SEQ ID NO:328, SEQ ID NO:346, SEQ IDNO:334, and SEQ ID NO:352 were provided as PCR products with Nde I andNot I restriction sites at the 5′ and 3′ ends, respectively. The PCRfragments were cloned into pCR-Blunt II-Topo (Invitrogen, Carlsbad,Calif.) as recommended by the manufacturer. The sequence was verified bysequencing (Agencourt, Beverly, Mass.) and an insert with the correctsequence was then released from the vector using Nde I and Not Irestriction enzymes and ligated into the Nde I and Not I restrictionsites of pET30a. One skilled in the art can synthesize the genesencoding these racemases using various published techniques for example,as described in Stemmer et al., supra.

pET30a constructs of all racemase candidates listed above weretransformed into the E. coli expression host BL21DE3. Liquid cultureswere grown overnight in LB medium (BD Diagnostics, Franklin Lakes, N.J.)containing 50 μg/ml kanamycin at 37° C. with agitation at 250 rpm. Theseovernight cultures were used to inoculate shake flasks containing 50 mLOvernight Express™ media (Solutions 1-6, Novagen/EMD Biosciences, SanDiego, Calif.) containing 50 μg/ml kanamycin. Overnight Express™cultures were grown at 30° C. with agitation at 250 rpm forapproximately 20 hours, and cells were harvested by centrifugation whenOD₆₀₀ reached ˜6-10.

Cell extracts were typically prepared from the above frozen pellets byadding 5 ml per g of cell pellet of Bugbuster Amine Free (Novagen/EMDBiosciences, San Diego, Calif.) with 5 μL/mL of Protease InhibitorCocktail II (Calbiochem, San Diego, Calif.) and 1 μl/ml of benzonasenuclease (Novagen/EMD Biosciences, San Diego, Calif.). Cell solutionswere incubated at room temperature with gentle mixing for 15 min; cellswere spun out at 14000 rpm for 20 min (at 4° C.) and the supernatant wascarefully removed. Detergents and low molecular weight molecules wereremoved by passage through PD-10 columns (GE Healthcare, Piscataway,N.J.) previously equilibrated with 100 mM potassium phosphate (pH 7.8)with 0.05 mM PLP. Proteins were eluted with 3.5 mL of the same buffer.Total protein concentration was determined using the Pierce BCA proteinassay with bovine serum albumin (BSA) as the standard, per themanufacturer's instructions (Pierce Biotechnology, Inc., Rockford,Ill.). The resulting cell-free extract was used for subsequent assays.

Desalted cell-free extracts were evaluated using tryptophan racemaseassays under the conditions described in Example 17, with purified SEQID NO:442 serving as a positive control. For the tryptophan racemaseassay, a total of 10 μg and 100 μg BAR equivalent SEQ ID NO:442 racemase(based on Pierce BCA total protein analysis with BSA as the standard andestimation of percentage of BAR protein expressed from Experion,(Experion, version A.01.10, Biorad, Hercules, Calif.)), were used aspositive controls. 1 mg of total protein was added for each BD racemasecandidate being tested (based on Pierce BCA total protein analysis withBSA as the standard). Formation of D-tryptophan was measured at 30minutes, 1 hour, 2 hours, and 4 hours as described in Example 18. In afollow up experiment, a 24-hour sample was evaluated for D-tryptophanproduction.

None of the racemases listed above showed detectable tryptophan racemaseactivity at 24 hours under the conditions described herein. Tryptophanracemase activity was seen for positive control SEQ ID NO:442. The hostorganisms, expression conditions, and post expression cell handling canall affect whether there is detectable tryptophan racemase activityunder the conditions of the assay. Additionally, under optimizedconditions, it is expected that all racemase candidates could havetryptophan racemase activity.

Second Group

Racemases SEQ ID NO:322, SEQ ID NO:324 and SEQ ID NO:348 were providedas PCR products with Nde I and Not I restriction sites at the 5′ and 3′ends, respectively. However all of these sequences had additional Nde Iand/or Not I sites internal to the gene sequence so direct subcloningwas not possible. SEQ ID NO:350 was re-amplified by PCR with RTthpolymerase (Applied Biosystems, Foster City, Calif.) and primers addingan Nde I and Xho I restriction site at the 5′ and 3′ ends, respectively.

Designation Sequence SEQ ID NO: SEQ IDTAAGAAGGAGATATACATATGGAATTCGATTGGATTCG 517 NO: 324 infusion F Nde1SEQ ID GGTGGTGGTGCTCGAGTGCGGCCGCTTATAACACCTG 518 NO: 324 infusion R Not1SEQ ID TAAGAAGGAGATATACATATGTCGCATTCCACCACCTGG 519 NO: 348 infusion FNde1 SEQ ID GGTGGTGGTGCTCGAGTGCGGCCGCTCAGCGATACTG 520 NO: 348 infusion RNot1 SEQ ID TAAGAAGGAGATATACATATGAAAAGTGCAGGCATTATA G 521 NO: 322infusion F Nde1 SEQ ID GGTGGTGGTGCTCGAGTGCGGCCGCTTAAGCCTTAGT 522 NO: 322infusion R Not1

The PCR fragment was digested with Nde I and Xho I restriction enzymesand ligated into the Nde I and Xho I restriction sites of pET30a.Correct plasmids were verified by digestion with Nde I and Xho I andsequencing (Agencourt, Beverly, Mass.). One skilled in the art cansynthesize the genes encoding these racemases using various publishedtechniques for example, as described in Stemmer et al., supra.

pET30a clones of all of the above racemases were transformed intoexpression host BL21DE3. Liquid cultures were grown overnight (LBkanamycin 50, 37° C., 250 rpm) and used to inoculate shake flaskscontaining 50 mL Overnight Express™ media (Solutions 1-6, Novagen/EMDBiosciences, San Diego, Calif.) containing kanamycin. Overnight Express™cultures were grown at 30° C. and 250 rpm for approximately 20 hours,and collected when the OD₆₀₀ reached ˜6-10. Cells were harvested bycentrifugation.

Desalted cell-free extracts of racemase candidates SEQ ID NO:322, SEQ IDNO:324, and SEQ ID NO:348 were prepared as described above.

Tryptophan racemase assays were carried out under the conditionsdescribed in Example 17, with purified A. caviae D76N BAR (see Example19) serving as a positive control. For the tryptophan racemase assay, atotal of 50 μg BAR equivalent of positive control (based on Pierce BCAtotal protein analysis with BSA as the standard and estimation ofpercentage of BAR protein expressed from Experion (Experion, versionA.01.10, Biorad, Hercules, Calif.) was added. 1 mg of total protein wasadded for each BD racemase candidate being tested (based on Pierce BCAtotal protein analysis with BSA as the standard). Formation ofD-tryptophan was measured at 1 hour, 2 hours, 4 hours, and 21.5 hours asdescribed in Example 18.

TABLE 33 D-trp production D-trp production, μg/mL Enzyme 1 hour 2 hours4 hours 21.5 hours A. caviae D76N pure 742.7 1368 2160.7 2458.7 SEQ IDNO: 322 305 437.7 596 1174.3 SEQ ID NO: 324 nd nd nd nd SEQ ID NO: 348nd nd nd nd nd = not detected under the conditions of the assay asdescribed above.

Tryptophan racemase activity was observed for SEQ ID NO:322. This enzymeis interesting because it is the smallest racemase protein that wasactive on tryptophan, with the protein being only 232 amino acids (ascompared to 409 amino acids for the A. caviae benchmark, and >300 aminoacids for most of the other racemase candidates).

There was no detectable tryptophan racemase activity observed for SEQ IDNO:324 and SEQ ID NO:348 under the conditions tested. SDS-PAGE analysisshowed good soluble protein expression for SEQ ID NO:348, but minimalsoluble protein expression for SEQ ID NO:324. The host organisms,expression conditions, and post expression cell handling can all affectwhether there is detectable tryptophan racemase activity under theconditions of the assay. Additionally, under optimized conditions, it isexpected that all racemase candidates could have tryptophan racemaseactivity.

Third Group

Racemases SEQ ID NO:340 and SEQ ID NO:350 were provided as PCR productswith Nde I and Not I restriction sites at the 5′ and 3′ ends,respectively. However, all of these sequences had additional Nde Iand/or Not I sites internal to the gene sequence so direct subcloningwas not carried out. SEQ ID NO:350 was re-amplified by PCR with RTthpolymerase (Applied Biosystems, Foster City, Calif.) and primers addingan Nde I and Xho I restriction site at the 5′ and 3′ ends, respectively.

SEQ ID Designation Sequences NO: SEQ ID NO: 350GGTTCCAACATATGACCGGGCTGACGGTAACGGCGGTG 523 FNde1 SEQ ID NO: 350GGTTCCAACTCGAGATAATAGCGGCCGCTCATCCC 524 RXho1 SEQ ID NO: 340TAAGAAGGAGATATACATATGAGACCAGCTCGCGTTAGC 525 infusion F Nde1SEQ ID NO: 340 GGTGGTGGTGCTCGAGTGCGGCCGCCTAGTTCCAGTT 526 infusion R Not1

The PCR fragment was digested with Nde I and Xho I restriction enzymesand ligated into the Nde I and Xho I restriction sites of pET30a.Correct plasmids were verified by digestion with Nde I and Xho I andsequencing (Agencourt, Beverly, Mass.). One skilled in the art cansynthesize the genes encoding these racemases using various publishedtechniques for example, as described in Stemmer et al., supra.

pET30a constructs of all racemase candidates listed above weretransformed into the E. coli expression host BL21DE3. Liquid cultureswere grown overnight in LB medium (BD Diagnostics, Franklin Lakes, N.J.)containing 50 μg/ml kanamycin at 37° C. with agitation at 250 rpm. Theseovernight cultures were used to inoculate shake flasks containing 50 mLOvernight Express™ media (Solutions 1-6, Novagen/EMD Biosciences, SanDiego, Calif.) containing 50 μg/ml kanamycin. Overnight Express™cultures were grown at 30° C., with agitation at 250 rpm forapproximately 20 hours, and cells were harvested by centrifugation whenOD₆₀₀ reached ˜6-10.

Desalted cell-free extracts of racemase candidates SEQ ID NO:340 and SEQID NO:350 were prepared as described above.

Tryptophan racemase assays were carried out under the conditionsdescribed in Example 17, with SEQ ID NO:412 serving as a positivecontrol.

For the tryptophan racemase assay, a total of ˜5 μg BAR equivalent ofcontrol (based on Pierce BCA total protein analysis with BSA as thestandard and estimation of percentage of BAR protein expressed fromExperion, (Experion, version A.01.10, Biorad, Hercules, Calif.) wasadded, and 1 mg of total protein was added for each racemase candidatebeing tested (based on Pierce BCA total protein analysis with BSA as thestandard). Formation of D-tryptophan was measured at 15 minutes, 2hours, and 21 hours as described in Example 18.

No tryptophan racemization was detected for SEQ ID NO:340 or SEQ IDNO:350 under the conditions tested. Positive control SEQ ID NO:412showed tryptophan racemase activity. SDS-PAGE analysis showed lowsoluble protein expression for SEQ ID NO:340 and SEQ ID NO:350.

The host organisms, expression conditions, and post expression cellhandling can all affect whether there is detectable tryptophan racemaseactivity under the conditions of the assay. Additionally, underoptimized conditions, it is expected that all racemase candidates couldhave tryptophan racemase activity.

Example 16 Analysis of Racemases Provided as PCR-4-Blunt TOPO Clones

Racemases SEQ ID NO:336, SEQ ID NO:338, SEQ ID NO:358, SEQ ID NO:360,SEQ ID NO:362, and SEQ ID NO:366 were provided as PCR-4-Blunt TOPOclones. Racemases in these plasmids were amplified with RTth polymerase(Applied Biosystems, Foster City, Calif.) and primers adding an Nde Iand Xho I restriction site at the 5′ and 3′ ends, respectively.

SEQ ID Clone Sequence NO: SEQ ID GGTTCCAAGGCATATGAGAACGAGACATCGATGCTG527 NO: 336FNde1 SEQ ID GGTTCCAATTCTCGAGTCAGGCTGCGTGACTGCCGCGA 528NO: 336RXho1 SEQ ID TTCCAATTGGCATATGCACCGCGTCTGGGCTGAAATC 529NO: 338FNde1 SEQ ID GGTTCCAACTCGAGTCACACATACACACGCGCCACGC 530NO: 338RXho1 SEQ ID TTGGAACCTTCATATGCAAACGAAACCCGCGCCCCG 531NO: 356FNde1 SEQ ID GGTTCCAAGGCTCGAGTTAGCGAATGTAAACCCGTTCCAC 532NO: 356RXho1 SEQ ID CCTTGGAACATATGGAACGAATCGTCCAGAAGCTGC 533NO: 358FNde1 * SEQ ID GGCCTTAACTCGAGTTATGACATCCGCGGAATCC 534NO: 358RXho1 * SEQ ID CCTTGGAACATATGGAACGAATCGTCCAGAAGCTGC 535NO: 360FNde1 * SEQ ID GGCCTTAACTCGAGTTATGACATCCGCGGAATCC 536NO: 360RXho1 * SEQ ID CCTTGGAACATATGGAACGAATCGTCCAGAAGCTGC 537NO: 362FNde1 * SEQ ID GGCCTTAACTCGAGTTATGACATCCGCGGAATCC 538NO:362RXho1 * SEQ ID CCTTGGAACATATGGAACGAATCGTCCAGAAGCTGC 539NO: 366FNde1 * SEQ ID GGCCTTAACTCGAGTTATGACATCCGCGGAATCC 540NO: 366RXho1 * * Same forward and reverse primer pair was used for SEQID NO: 358, SEQ ID NO: 360, SEQ ID NO: 362, and SEQ ID NO: 366 due to100% DNA homology in primer regions.

The PCR fragments were cloned into pCR-Blunt II-Topo (Invitrogen,Carlsbad, Calif.) as recommended by the manufacturer. The sequence wasverified by sequencing (Agencourt, Beverly, Mass.) and an insert withthe correct sequence was then released from the vector using Nde I andXho I restriction enzymes (New England Biolabs, Ipswich, Mass.) andligated into the Nde I and Xho I restriction sites of pET30a. See Tableabove for specific primers and plasmids names. (It is noted that theTOPO cloning efforts for SEQ ID NO:356 were unsuccessful after multipleattempts, so this racemase was not further processed). One skilled inthe art can synthesize the genes encoding these racemases using variouspublished techniques for example, as described in Stemmer et al., supra.

pET30a constructs of all racemase candidates listed above weretransformed into the E. coli expression host BL21DE3. Liquid cultureswere grown overnight in LB medium (BD Diagnostics, Franklin Lakes, N.J.)containing 50 μg/ml kanamycin at 37° C. with agitation at 250 rpm. Theseovernight cultures were used to inoculate shake flasks containing 50 mLOvernight Express™ media (Solutions 1-6, Novagen/EMD Biosciences, SanDiego, Calif.) containing 50 μg/ml kanamycin. Overnight Express™cultures were grown at 30° C. with agitation at 250 rpm forapproximately 20 hours, and cells were harvested by centrifugation whenOD₆₀₀ reached ˜6-10.

Desalted cell-free extracts of batch 8 racemases SEQ ID NO:336, SEQ IDNO:338, SEQ ID NO:358, SEQ ID NO:360, SEQ ID NO:362, and SEQ ID NO:366were prepared as described below (SEQ ID NO:356 and SEQ ID NO:364 fromthis experiment were not characterized).

Cell extracts were typically prepared from the above frozen pellets byadding 5 ml per g of cell pellet of Bugbuster Amine Free (Novagen/EMDBiosciences, San Diego, Calif.) with 5 μL/mL of Protease InhibitorCocktail II (Calbiochem, San Diego, Calif.) and 1 μl/ml of benzonasenuclease (Novagen/EMD Biosciences, San Diego, Calif.). Cell solutionswere incubated at room temperature with gentle mixing for 15 min; cellswere spun out at 14000 rpm for 20 min (at 4° C.) and the supernatant wascarefully removed. Detergents and low molecular weight molecules wereremoved by passage through PD-10 columns (GE Healthcare, Piscataway,N.J.) previously equilibrated with 100 mM potassium phosphate (pH 7.8)with 0.05 mM PLP. Proteins were eluted with 3.5 mL of the same buffer.Total protein concentration was determined using the Pierce BCA (PierceBiotechnology, Inc., Rockford, Ill.) protein assay with bovine serumalbumin (BSA) as the standard, per the manufacturer's instructions. Theresulting cell-free extract was used for subsequent assays.

Tryptophan racemase assays were carried out under the conditionsdescribed in Example 17, with purified A. caviae D76N BAR (Example 19)serving as a positive control. A total of 100 μg BAR equivalent ofcontrol was added (based on Pierce BCA total protein analysis with BSAas the standard and estimation of percentage of BAR protein expressedfrom Experion, version A.01.10, Biorad, Hercules, Calif.), and 1 mg oftotal protein was added for each racemase candidate being tested.Formation of D-tryptophan was measured at 30 minutes, 2 hours, 4 hoursand 52 hours as described in Example 18.

TABLE 34 D-trp production D-tryptophan production, μg/mL Enzyme 30 min 2hours 4 hours 52 hours SEQ ID NO: 336 1485 2787 2705 2434 SEQ ID NO: 3381503 2757 2738 2353 SEQ ID NO: 366 nd nd nd nd SEQ ID NO: 358 nd nd nd27 SEQ ID NO: 360 nd nd nd nd SEQ ID NO: 362 nd nd nd nd SEQ ID NO: 442 340 1020 1726 3011 purified - 100 μg A. caviae D76N 1036 2804 2849 3011purified - 100 μg nd = not detected under the conditions of the assay asdescribed above.

Racemase candidates SEQ ID NO:336, SEQ ID NO:338 and SEQ ID NO:358 wereactive. Racemase candidates SEQ ID NO:366, SEQ ID NO:360, and SEQ IDNO:362 showed no detectable tryptophan racemase activity under theconditions tested. SEQ ID NO:366, SEQ ID NO:360, and SEQ ID NO:362 allhad satisfactory soluble protein expression. The host organisms,expression conditions, and post expression cell handling can all affectwhether there is detectable tryptophan racemase activity under theconditions of the assay. Additionally, under optimized conditions, it isexpected that all racemase candidates could have tryptophan racemaseactivity.

Example 17 Description of Racemase Assay Conditions

Leucine, Phenylalanine, Tryptophan, Methionine, Tyrosine, Alanine,Lysine, Aspartic Acid, Glutamate Racemase Assay

Racemase assays were performed starting with the L-amino acid isomer andthe formation of corresponding D-amino acid was followed.

Assay Conditions:

30 mM L-amino acid (L-Leucine, L-Phenylalanine, L-Tryptophan,L-Methionine, L-Tyrosine, L-Alanine, L-Lysine, L-Aspartic Acid, orL-Glutamate), 50 mM Potassium phosphate buffer (pH 8.0), 0.05 mM PLP,and water was added to make the volume up to 1 mL.

The assays were conducted at 30° C. with shaking at 225 rpm. Desaltedracemase candidate proteins (cell-free extracts or purifiedpreparations) were evaluated for amino acid racemase activity. Whereverpossible, appropriate negative and positive controls were included forthe assays. Sample aliquots were taken for analysis at varioustimepoints and formic acid was added to a final concentration of 2% tostop the reaction. Samples were frozen at −80° C., then thawed,centrifuged and filtered through 0.2μ filter (Pall Life Sciences, AnnArbor, Mich.). Samples were analyzed for D-amino acid using the chiralLC/MS/MS method described in Example 18.

Monatin Racemase Assay

A subset of racemase candidates that gave promising tryptophan racemaseresults was tested for monatin racemization.

Assay Conditions:

10 mM R,R monatin, 50 mM Potassium phosphate buffer (pH 8.0), 0.05 mMPLP, and water were added to make the volume up to 1 mL.

The assays were performed at 30° C. with shaking at 225 rpm. At varioustime points, sample aliquots were taken, diluted five-fold withdistilled water, then filtered through 0.2μ filter (Pall Life Sciences,Ann Arbor, Mich.) and stored at −80° C. for subsequent analysis. Sampleswere analyzed for the distribution of monatin stereoisomers as describedin Example 18.

Example 18 Detection of Monatin Stereoisomers and Chiral Detection ofLysine, Alanine, Methionine, Tyrosine, Leucine, Phenylalanine,Tryptophan, Glutamate, and Aspartate

This example describes methods used to detect the presence ofstereoisomers of monatin, lysine, alanine, methionine, tyrosine,leucine, phenylalanine, tryptophan, glutamate, and aspartate. It alsodescribes a method for the separation and detection of the fourstereoisomers of monatin.

Chiral LC/MS/MS (“MRM”) Measurement of Monatin

Determination of the stereoisomer distribution of monatin in in vitroreactions was accomplished by derivatization with1-fluoro-2-4-dinitrophenyl-5-L-alanine amide (“FDAA”), followed byreversed-phase LC/MS/MS MRM measurement.

Derivatization of Monatin with FDAA

To 50 μL of sample or standard and 10 μL of internal standard was added100 μL of a 1% solution of FDAA in acetone. Twenty μL of 1.0 M sodiumbicarbonate was added, and the mixture incubated for 1 h at 40° C. withoccasional mixing. The sample was removed and cooled, and neutralizedwith 20 μL of 2.0 M HCl (more HCl may be required to effectneutralization of a buffered biological mixture). After degassing wascomplete, samples were ready for analysis by LC/MS/MS.

LC/MS/MS Multiple Reaction Monitoring for the Determination of theStereoisomer Distribution of Monatin

Analyses were performed using the LC/MS/MS instrumentation describedabove. LC separations capable of separating all four stereoisomers ofmonatin (specifically FDAA-monatin) were performed on a Phenomenex Luna2.0×250 mm (3 μm) C18 (2) reversed phase chromatography column at 40° C.The LC mobile phase consisted of A) water containing 0.05% (mass/volume)ammonium acetate and B) acetonitrile. The elution was isocratic at 13%B, 0-2 min, linear from 13% B to 30% B, 2-15 min, linear from 30% B to80% B, 15-16 min, isocratic at 80% B 16-21 min, and linear from 80% B to13% B, 21-22 min, with an 8 min re-equilibration period between runs.The flow rate was 0.23 mL/min, and PDA absorbance was monitored from 200nm to 400 nm. All parameters of the ESI-MS were optimized and selectedbased on generation of deprotonated molecular ions ([M-H]−) ofFDAA-monatin, and production of characteristic fragment ions.

The following instrumental parameters were used for LC/MS analysis ofmonatin in the negative ion ESI/MS mode: Capillary: 3.0 kV; Cone: 40 V;Hex 1: 15 V; Aperture: 0.1 V; Hex 2: 0.1 V; Source temperature: 120° C.;Desolvation temperature: 350° C.; Desolvation gas: 662 L/h; Cone gas: 42L/h; Low mass resolution (Q1): 14.0; High mass resolution (Q1): 15.0;Ion energy: 0.5; Entrance: 0V; Collision Energy: 20; Exit: 0V; Low massresolution (Q2): 15; High mass resolution (Q2): 14; Ion energy (Q2):2.0; Multiplier: 650. Three FDAA-monatin-specific parent-to daughtertransitions are used to specifically detect FDAA-monatin in in vitro andin vivo reactions. The transitions monitored for monatin are 542.97 to267.94, 542.97 to 499.07, and 542.97 to 525.04. Monatin internalstandard derivative mass transition monitored was 548.2 to 530.2.Identification of FDAA-monatin stereoisomers is based on chromatographicretention time as compared to purified synthetic monatin stereoisomers,and mass spectral data. An internal standard wais used to monitor theprogress of the reaction and for confirmation of retention time of theS,S stereoisomer.

Detection of L- and D-Amino Acids by LC/MS/MS

Samples containing a mixture of L- and D-amino acids such as lysine,alanine, methionine, tyrosine, leucine, phenylalanine, tryptophan,glutamate, and aspartate from biochemical reaction experiments werefirst treated with formic acid to denature protein. The sample was thencentrifuged and filtered through a 0.2 μm nylon syringe filter prior toLC/MS/MS analysis. Identification of L- and D-amino acids was based onretention time and mass selective detection. LC separation wasaccomplished by using Waters 2690 liquid chromatography system and anASTEC 2.1 mm×250 mm Chirobiotic TAG chromatography column with columntemperature set at 45° C. LC mobile phase A and B were 0.25% acetic acidand 0.25% acetic acid in methanol, respectively. Isocratic elution wasused for all methods to separate the L and D isomers. Lysine was elutedusing 80% mobile phase A, and 20% B and a flow rate of 0.25 mL/minGlutamate, alanine, and methionine were separated with elution of 60%mobile phase A and 40% B and a flow rate of 0.25 mL/min Aspartate,tryptophan, tyrosine, leucine, and phenylalanine were separatedisomerically with 30% mobile phase A and 70% B with a flow rate of 0.3mL/min for aspartate and tryptophan, and 0.25 mL/min for tyrosine,leucine, and phenylalanine.

The detection system for analysis of L- and D-amino acids included aWaters 996 Photo-Diode Array (PDA) detector and a Micromass QuattroUltima triple quadrupole mass spectrometer. The PDA, scanning from 195to 350 nm, was placed in series between the chromatography system andthe mass spectrometer. Parameters for the MICROMASS QUATTRO ULTIMA™triple quadrupole mass spectrometer operating in positive electrosprayionization mode (+ESI) were set as the following: Capillary: 3.2 kV;Cone: 20 V; Hex 1: 12 V; Aperture: 0.1 V; Hex 2: 0.2V; Sourcetemperature: 120° C.; Desolvation temperature: 350° C.; Desolvation gas:641 L/h; Cone gas: 39 L/h; Low mass Q1 resolution: 16.0; High mass Q1resolution: 16.0; Ion energy 1: 0.1; Entrance: −5; Collision: 20; Exit1: 10; Low mass Q2 resolution: 16.0; High mass Q2 resolution: 16.0 Ionenergy 2: 1.0; Multiplier: 650 V. MS/MS experiments with MultipleReaction Monitoring (MRM) mode were set up to selectively monitorreaction transitions of 147.8 to 84.03, 147.8 to 56.3, and 147.8 to102.2 for glutamate, 133.85 to 74.03, 133.85 to 69.94 and 133.85 to87.99 for aspartate, 146.89 to 84.09, 146.89 to 55.97 and 146.89 to67.23 for lysine, 149.80 to 56.1, 149.8 to 61.01, and 149.80 to 104.15for methionine, 181.95 to 135.97, 181.95 to 90.88 and 181.95 to 118.87for tyrosine, 131.81 to 86.04 and 131.81 to 69.31 for leucine, 90.0 to44.3 for alanine, and 165.83 to 102.96, 165.83 to 93.27 and 165.83 to120.06 for phenylalanine. In the case where numerous transitions arelisted, the first transition listed was used for quantification. Fortryptophan, MS/MS experiments with Multiple Reaction Monitoring (MRM)mode were set up to selectively monitor reaction transitions of 205.0 to145.91, 205.0 to 117.92, and 205.0 to 188.05, and the transition from212.0 to 150.98 for d8-DL tryptophan. Tryptophan quantification wasachieved by determining the ratio of analyte response of transition205.0 to 145.91 to that of the internal standard, d8-D,L tryptophan.

Production of Monatin for Standards and for Assays

A racemic mixture of R,R and S,S monatin was synthetically produced asdescribed in U.S. Pat. No. 5,128,482.

The R,R and S,S monatin were separated by a derivatization andhydrolysis step. Briefly, the monatin racemic mixture was esterified,the free amino group was blocked with Cbz, a lactone was formed, and theS,S lactone was selectively hydrolyzed using an immobilized proteaseenzyme. The monatin can also be separated as described in Bassoli etal., 2005, Eur. J. Org. Chem., 8:1652-1658.

Example 19 Cloning and Analysis of Broad Activity Racemase (BAR) fromAeromonas caviae ATCC 14486

Since tryptophan racemase activity was detected in crude extracts fromAeromonas caviae ATCC 14486, degenerate primers were designed (based onconserved regions of known BAR homologs) to obtain the BAR gene fromAeromonas caviae ATCC 14486. Degenerate primer sequences are shownbelow:

Aer deg F2: (SEQ ID NO: 541) 5′-GCCAGCAACGARGARGCMCGCGT-3′; andAer deg R1: (SEQ ID NO: 542) 5′-TGGCCSTKGATCAGCACA-3′

wherein K indicates G or T, R indicates A or G, S indicates C or G, andM indicates A or C.

The above primers were used to PCR amplify a 715 bp DNA fragment from A.caviae (ATCC Accession No. 14486) genomic DNA. The following PCRprotocol was used: A 50 μL reaction contained 0.5 μL template (˜100 ngof A. caviae genomic DNA), 1.6 μM of each primer, 0.3 mM each dNTP, 10 UrTth Polymerase XL (Applied Biosystems, Foster City, Calif.), 1×XLbuffer, 1 mM Mg(OAc)₂ and 2.5 μL dimethyl sulfoxide. The thermocyclerprogram used included a hot start at 94° C. for 3 minutes and 30repetitions of the following steps: 94° C. for 30 seconds, 53° C. for 30seconds, and 68° C. for 2 minutes. After the 30 repetitions, the samplewas maintained at 68° C. for 7 minutes and then stored at 4° C. This PCRprotocol produced a product of 715 bp.

The PCR product was gel purified from 0.8% TAE-agarose gel using theQiagen gel extraction kit (Qiagen, Valencia, Calif.). The product wasTOPO cloned and transformed into TOP 10 cells according tomanufacturer's protocol (Invitrogen, Carlsbad, Calif.). The plasmid DNAwas purified from the resulting transformants using the Qiagen spinminiprep kit (Qiagen, Valencia, Calif.) and screened for the correctinserts by restriction digest with EcoR 1. The sequences of plasmidsappearing to have the correct insert were verified by dideoxy chaintermination DNA sequencing with universal M13 forward primers.

Four libraries were constructed for each strain as per manufacturer'sprotocols (BD GenomeWalker™ Universal Kit, Clontech). Gene-specificprimers were designed as per GenomeWalker™ manufacturer's protocolsbased on sequences obtained using degenerate primer sequences (seeabove), allowing for a few hundred homologous base pair overlap withoriginal product. These gene-specific primers were subsequently usedwith GenomeWalker™ adaptor primers for PCR of upstream and downstreamsequences to complete A. caviae BAR ORE.

Full-Length Gene Sequence of the A. caviae BAR Gene

(SEQ ID NO: 543) atgcacaaga aaacactgct cgcgaccctg atctttggcctgctggccgg ccaggcagtc gccgccccct atctgccgctcgccgacgac caccgcaacg gtcaggaaca gaccgccgccaacgcctggc tggaagtgga tctcggcgcc ttcgagcacaacatccagac cctgaagaat cgcctcggtg acaagggcccgcagatctgc gccatcatga aggcggacgc ctacggtcacggcatcgacc tgctggtccc ttccgtggtc aaggcaggcatcccctgcat cggcatcgcc agcaacgaag aagcacgtgttgcccgcgag aagggcttcg aaggtcgcct gatgcgggtacgtgccgcca ccccggatga agtggagcag gccctgccctacaagctgga ggagctcatc ggcagcctgg agagcgccaaggggatcgcc gacatcgccc agcgccatca caccaacatcccggtgcaca tcggcctgaa ctccgccggc atgagccgcaacggcatcga tctgcgccag gacgatgcca aggccgatgccctggccatg ctcaagctca aggggatcac cccggtcggcatcatgaccc acttcccggt ggaggagaaa gaggacgtcaagctggggct ggcccagttc aagctggact accagtggctcatcgacgcc ggcaagctgg atcgcagcaa gctcaccatccacgccgcca actccttcgc caccctggaa gtaccggaagcctactttga catggtgcgc ccgggcggca tcatctatggcgacaccatt ccctcctaca ccgagtacaa gaaggtgatggcgttcaaga cccaggtcgc ctccgtcaac cactacccggcgggcaacac cgtcggctat gaccgcacct tcaccctcaagcgcgactcc ctgctggcca acctgccgat gggctactccgacggctacc gccgcgccat gagcaacaag gcctatgtgctgatccatgg ccagaaggcc cccgtcgtgg gcaagacttccatgaacacc accatggtgg acgtcaccga catcaaggggatcaaacccg gtgacgaggt ggtcctgttc ggacgccagggtgatgccga ggtgaaacaa tctgatctgg aggagtacaacggtgccctc ttggcggaca tgtacaccgt ctggggctataccaacccca agaagatcaa gcgctaa.The Corresponding Amino Acid Sequence for the A. caviae Native BAR

(SEQID NO: 544) MHKKTLLATL IFGLLAGQAV AAPYLPLADD HRNGQEQTAANAWLEVDLGA FEHNIQTLKN RLGDKGPQIC AIMKADAYGHGIDLLVPSVV KAGIPCIGIA SNEEARVARE KGFEGRLMRVRAATPDEVEQ ALPYKLEELI GSLESAKGIA DIAQRHHTNIPVHIGLNSAG MSRNGIDLRQ DDAKADALAM LKLKGITPVGIMTHFPVEEK EDVKLGLAQF KLDYQWLIDA GKLDRSKLTIHAANSFATLE VPEAYFDMVR PGGIIYGDTI PSYTEYKKVMAFKTQVASVN HYPAGNTVGY DRTFTLKRDS LLANLPMGYSDGYRRAMSNK AYVLIHGQKA PVVGKTSMNT TMVDVTDIKGIKPGDEVVLF GRQGDAEVKQ SDLEEYNGAL LADMYTVWGY TNPKKIKR.

The following PCR primers were utilized to clone the native full-lengthA. caviae BAR in both tagged and C-terminally his-tagged versions:

A. caviae F Nde1 (SEQ ID NO: 545)5′-GGA ACC TTC ATA TGC ACA AGA AAA CAC TGC TCG CGA CC-3′;A. caviae R BamH1 (untagged) (SEQ ID NO: 546)5′-GGT TCC AAG GAT CCT TAG CGC TTG ATC TTC TTG GGG TTG-3′; andA. caviae R Xho1 (C-term tag) (SEQ ID NO: 547)5′-TTC CAA GGC TCG AGG CGC TTG ATC TTC TTG GGG TTG GT-3′.

The C-terminally tagged enzyme had comparable activity to the untaggednative A. caviae BAR. When 200 μg of purified (tagged) racemase enzymeswere used in a tryptophan racemase assay as described in Example 17, at30 minutes, A. caviae BAR produced 1034 μg/mL of D-tryptophan.

The first 21 N-terminal amino acid residues of the A. caviae native BARamino acid sequence above (SEQ ID NO:544) were predicted to be a signalpeptide using the program Signal P 3.0 ((cbs.dtu.dk/services/SignalP/ onthe World Wide Web). The following N-terminal primer was used to clonethe A. caviae gene without amino acids 2-21 of the leader sequence:A.cavMinus leader F NdeI 5′ CCT TGG AAC ATA TGG CCC CCT ATC TGC CGC T 3′(SEQ ID NO:548).

The leaderless racemase, when expressed, was found to retainapproximately 65% of the activity, as compared with the expressionproduct of the full-length gene. The periplasmic and cytoplasmic proteinfractions were isolated for the wild type expression products, as wellas the leaderless constructs, as described in the pET System Manual(Novagen, Madison, Wis.). The majority of expressed wildtype BAR wasfound in the periplasm, while the leaderless BAR appeared to remain inthe cytoplasm. The reduction in activity of the leaderless A. caviae BARmay be due to a change in processing and/or folding when expressed inthe cytoplasm.

Effect of Leader Sequence on Racemase

A D76N mutant of A. caviae BAR was made to determine if this positionwas critical for broad activity. Mutagenesis was done using theQuickChange-Multi site-directed mutagenesis kit (Stratagene, La Jolla,Calif.), using the C-tagged A. caviae BAR gene in pET30 as template. Thefollowing mutagenic primer was used to make a D76N change (nucleotideposition 226): 5′-CGC CAT CAT GAA GGC GAA CGC CTA CGG TCA CG-3′ (SEQ IDNO:549). The site-directed mutagenesis was done as described in themanufacturer's protocol. The mutant and the wildtype enzyme wereproduced as described above and assayed as described in Example 17 using200 micrograms of purified protein (prepared as describedherein—purified A. caviae D76N was C-term His tagged in pET30) andapproximately 7 mg/mL of L-tryptophan as substrate. At the 30 minutetime point, the mutant produced 1929 micrograms per mL of D-tryptophanas compared to 1149 micrograms per mL for the wildtype enzyme. The D76Nmutant also reached equilibrium at an earlier time point. Theimprovement in activity was unexpected.

Based on the high homology in this region for Aeromonas and PseudomonasBAR enzymes, it might be expected that similar mutations in other broadactivity racemases would also be beneficial. A benefit effect, however,was not observed when a similar mutation in SEQ ID NO:442 was generated.See Example 13.

The following racemase candidates had 99% identity at the amino acidlevel to the BAR from A. caviae described in this example: SEQ IDNO:200, SEQ ID NO:202, SEQ ID NO:206, SEQ ID NO:142, SEQ ID NO:186 andSEQ ID NO:176. SEQ ID NO:176 had 97% identity at the amino acid level tothe BAR from A. caviae described in this example. It is expected thatthese candidates would also have tryptophan racemase activity given thehigh sequence homology to an enzyme with demonstrated tryptophanracemase activity.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. An expression cassette, a vector, or a cloningvehicle comprising an isolated, synthetic or recombinant polynucleotidethat encodes a polypeptide having racemase activity, wherein thepolynucleotide is selected from the group consisting of: (a) a nucleicacid-sequence having at least 95%, 96%, 97%, 98%, or 99%, sequenceidentity to the full length nucleic acid sequence of SEQ ID NO: 411, (b)a nucleic acid sequence that hybridizes under stringent conditions tofull length nucleic acid sequence of SEQ ID NO:411 and the stringentconditions comprise a wash step comprising a wash in 0.2X SSC at atemperature of about 65° C. for about 15 minutes; (c) the nucleic acidsequence of SEQ ID NO:411; and (d) a nucleic acid sequence fullycomplementary to the sequence of any of (a) to (c).
 2. The expressioncassette, a vector, or a cloning vehicle of claim 1, wherein the cloningvehicle comprises a viral vector, a plasmid, a phage, a phagemid, acosmid, a fosmid, a bacteriophage or an artificial chromosome.
 3. Thecloning vehicle of claim 2, wherein the viral vector comprises anadenovirus vector, a retroviral vector or an adeno-associated viralvector, or, the artificial chromosome comprises a bacterial artificialchromosome (BAC), a bacteriophage P1-derived vector (PAC), a yeastartificial chromosome (YAC), or a mammalian artificial chromosome (MAC).4. An isolated host cell comprising the polynucleotide of claim 1,wherein the host cell is a bacterial cell, a mammalian cell, a fungalcell, a yeast cell, an insect cell or a plant cell.
 5. A method ofproducing a recombinant polypeptide comprising the steps of: (a)providing the polynucleotide of claim 1, operably linked to a promoter;and (b) expressing the polynucleotide of step (a) under conditions thatallow expression of the polypeptide, thereby producing a recombinantpolypeptide, and optionally the method further comprises transforming anisolated host cell with the polynucleotide of step (a) followed byexpressing the nucleic acid of step (a), thereby producing a recombinantpolypeptide in a transformed cell.
 6. A method of generating a variantof the polynucleotide of claim 1 comprising the steps of: providing atemplate nucleic acid comprising the sequence of claim 1; and modifying,deleting, or adding one or more nucleotides in the template sequence, ora combination thereof, to generate a variant of the template nucleicacid.
 7. The polynucleotide of claim 1, and encoding a variantpolypeptide having at least one conservative amino acid substitution andthe variant retains racemase activity.
 8. The nucleic acid of claim 7,wherein the at least one conservative amino acid substitution isselected from: (a) substituting an amino acid with another amino acid oflike characteristics; (b) replacement of an aliphatic amino acid withanother aliphatic amino acid; (c) replacement of a Serine with aThreonine or vice versa; (d) replacement of an acidic residue withanother acidic residue; (e) replacement of a residue bearing an amidegroup with another residue bearing an amide group; (f) exchange of abasic residue with another basic residue; and (g) replacement of anaromatic residue with another aromatic residue.
 9. The polynucleotide ofclaim 1, encoding a polypeptide having racemase activity but lacking asignal sequence, or a prepro domain.
 10. The polynucleotide of claim 1,encoding a polypeptide having racemase activity, and further comprisinga heterologous sequence.
 11. The polynucleotide of claim 10, wherein theheterologous sequence is a heterologous domain, a heterologous catalyticdomain (CD), or a combination thereof.
 12. The polynucleotide of claim11, wherein the heterologous signal sequence, domain or catalytic domain(CD) is derived from a heterologous enzyme.
 13. The polynucleotide ofclaim 10, wherein the heterologous sequence is a tag, an epitope, atargeting peptide, a cleavable sequence, a detectable moiety or anenzyme.